WO2021194391A1 - Procédé d'analyse in vitro de sensibilité aux antibiotiques de bactéries et de champignons - Google Patents

Procédé d'analyse in vitro de sensibilité aux antibiotiques de bactéries et de champignons Download PDF

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Publication number
WO2021194391A1
WO2021194391A1 PCT/RU2021/050064 RU2021050064W WO2021194391A1 WO 2021194391 A1 WO2021194391 A1 WO 2021194391A1 RU 2021050064 W RU2021050064 W RU 2021050064W WO 2021194391 A1 WO2021194391 A1 WO 2021194391A1
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Prior art keywords
antibiotic
blood
sensitivity
presepsin
bacteria
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PCT/RU2021/050064
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English (en)
Russian (ru)
Inventor
Алексей Юрьевич ЯКОВЛЕВ
Мария Алексеевна РЯБИКОВА
Original Assignee
Алексей Юрьевич ЯКОВЛЕВ
Мария Алексеевна РЯБИКОВА
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Application filed by Алексей Юрьевич ЯКОВЛЕВ, Мария Алексеевна РЯБИКОВА filed Critical Алексей Юрьевич ЯКОВЛЕВ
Publication of WO2021194391A1 publication Critical patent/WO2021194391A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention relates to medicine, namely to clinical and laboratory diagnostics, and may be applicable, for example, for express analysis of the sensitivity of pathogenic microflora to prescribed antibiotic therapy for sepsis.
  • Closest to the claimed invention is a method for in vitro rapid analysis of antibiotic sensitivity of gram-negative bacteria in a patient (7), comprising taking a sample of a biological fluid containing gram-negative bacteria; Determination of the minimum concentration of total endotoxin (total LPS) by the method of activated particles by adding 100 ⁇ l (1%) buffer solution, 100 ⁇ l of biological fluid and 20 ⁇ l of MAC diagnosticum - Endotox spp.
  • the objective of the present invention is to expand the arsenal of methods for rapid in vitro analysis of antibiotic susceptibility of bacteria and fungi in a patient.
  • the technical result is a quick determination of the sensitivity of bacterial and fungal infections to antibiotics.
  • the technical result is achieved by the fact that in the method of express in vitro analysis of the sensitivity to antibiotics of bacteria and fungi from the patient, blood is taken from the patient, the mixture of blood with an antibiotic solution is incubated at a temperature of 37 ° C for 30 minutes, the content of the analyte in the blood is determined , compare the results of the study and determine the sensitivity of bacteria to the antibiotic.
  • blood is taken in an amount of 1 ml into two sterile tubes with an anticoagulant, then 50 ⁇ l of 0.9% sterile NaCl solution is injected into the first tube, and 50 ⁇ l of a freshly prepared solution of a single dose of an antibiotic diluted in 100 ml of a solvent for an antibiotic into the second tube, presepsin is used as an analyte, its content in the blood of the first and second test tubes is determined using a diagnostic kit on an immunochemiluminescent analyzer, and the sensitivity of bacteria and fungi to an antibiotic is determined by a decrease in the presepsin content in the second test tube by 10% or more in comparison with the presepsin content in the first test tube.
  • Presepsin (sCD14-ST) is a fragment of the CD 14 glycoprotein after the interaction of macrophages with gram-positive, gram-negative bacteria and fungi (8). Presepsin is widely used to diagnose sepsis (9). Presepsin levels are significantly higher in patients with sepsis than in patients with SIRS (systemic inflammatory response syndrome) or in apparently healthy people (10,11).
  • Antibacterial and antimycotic drugs cause either the death of the microorganism or the cessation of its reproduction, which is manifested in the bactericidal or bacteriostatic mechanism of their action.
  • the termination of the reproduction of microorganisms or their death reduces the production of presepsin by macrophages.
  • the invention is carried out as follows.
  • 1 ml of test blood is taken into 2 tubes with an anticoagulant, preferably with EDTA or with heparin. Then, samples are prepared to determine the content of presepsin in them.
  • an anticoagulant preferably with EDTA or with heparin.
  • samples are prepared to determine the content of presepsin in them.
  • the 1st test tube which serves as a control, add 50 ⁇ l of 0.9% sterile NaCl solution, and in the 2nd tube - 50 ⁇ l of a freshly prepared solution of a single dose of an antibacterial or antifungal drug diluted in 100 ml of sterile NaCl solution or in a special solvent, intended for a drug planned for empiric antibacterial or antimycotic therapy.
  • antibiotics to which bacteria and fungi are susceptible can be used as investigational drugs, in particular vancomycin, linezolid, tecoplanin, meropenem, imepenem, doripenem, ertapenem, gentamicin, amikacin, ampicillin / sulbactam, cefaperazone / sulbactam, cefteploxinef , ciproflaxacin, moxifloxacin, ofloxacin, levoflaxacin, polymyxin B, amoxicillin / clavunate, lincomycin, cefuroxime, rifampicin, tobramycin, azithromycin, clarithromycin, fluconazole, ketoconazole, intraconazole, voriconazole, caspofungin, and other antibacterial and antimycotic drugs.
  • the content of presepsin in the blood of the 1st and 2nd test tubes is determined.
  • the content of presepsin is determined using a diagnosticum (test system) on an immunochemiluminescence analyzer.
  • the PATHFAST TM Presepsin diagnostic is used for in vitro diagnostics on the PATHFAST TM analyzer.
  • the analysis procedure is based on the method of chemiluminescence enzyme immunoassay.
  • Diagnosticum is a cartridge in which all the components necessary for testing are packed.
  • a reagent cartridge consists of wells filled with test reagents into which samples are added. After loading the cartridge into the diagnostic analyzer, a quantitative result can be obtained in 17 minutes.
  • polyclonal antibodies to presepsin associated with alkaline phosphatase and monoclonal antibodies to presepsin on magnetic particles are mixed with the sample. Presepsin from the sample binds to antibodies to it, forming an immunocomplex with enzyme-labeled antibodies and antibodies on magnetic particles.
  • a chemiluminescent substrate is added to the immune complex. After a short incubation, under the influence of an enzymatic reaction, luminescence begins in the mixture, the intensity of which depends on the concentration of presepsin in the sample. The result is calculated using a standard calibration curve.
  • a decrease in the content of presepsin in the patient's blood by 10% or more relative to the values in the 1st (control) test tube is determined as the sensitivity of the microorganism or fungal pathogen to the planned use of an antibacterial or antimycotic drug in a patient with sepsis.
  • the criterion for the sensitivity of bacteria or fungi to the test drug is an in vitro decrease in the content of presepsin in the patient's blood by 10% or more relative to the values in the control.
  • Example 1 Patient M. 35 years old. Diagnosis: condition of cesarean section. Purulent metroendometritis. Sepsis. Operation: extirpation of the uterus with appendages, sanitation and drainage of the abdominal cavity.
  • Example 2 Patient N., 58 years old. Diagnosis: Viral and bacterial pneumonia. Sepsis. Multiple organ failure. Upon admission to the hospital, the patient's blood was taken for bacteriological examination and conducting an antibiotic susceptibility test for levofloxacin. When levofloxacin was added to the blood after 30 minutes, the content of presepsin in the 1st test tube (control) was 1429 pkg / ml, and in the second (with the addition of levofloxacin) - 1205 pkg / ml. Antibacterial therapy with levofloxacin was started within 1 hour after admission to the hospital
  • Example 3 Patient F., 33 years old. Diagnosis: Acute myeloid leukemia, first attack. Nosocomial pneumonia. Fungal sepsis.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte au domaine de la médecine, concerne notamment un diagnostic clinique en laboratoire, et peut par exemple être utilisée pour l'analyse express de la sensibilité d'une microflore pathogène envers une thérapie antibactérienne ciblée dans le cas de septicémie. L'invention permet de déterminer rapidement la sensibilité d'une infection bactérienne et fongique envers des antibiotiques.
PCT/RU2021/050064 2020-03-27 2021-03-12 Procédé d'analyse in vitro de sensibilité aux antibiotiques de bactéries et de champignons WO2021194391A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2020112640 2020-03-27
RU2020112640A RU2735983C1 (ru) 2020-03-27 2020-03-27 Способ экспресс-анализа in vitro чувствительности к антибиотикам бактерий и грибов у пациента

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WO2021194391A1 true WO2021194391A1 (fr) 2021-09-30

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015115926A1 (fr) * 2014-01-31 2015-08-06 Общество С Ограниченной Ответственностью Научно-Производственная Фирма "Рохат" (Ооо Нпф "Рохат") Procédé d'analyse express in vitro de la sensibilité de bactéries gram-négatives à des antibiotiques et utilisation pour contrôler l'efficacité d'une thérapie antibactérienne

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015115926A1 (fr) * 2014-01-31 2015-08-06 Общество С Ограниченной Ответственностью Научно-Производственная Фирма "Рохат" (Ооо Нпф "Рохат") Procédé d'analyse express in vitro de la sensibilité de bactéries gram-négatives à des antibiotiques et utilisation pour contrôler l'efficacité d'une thérapie antibactérienne

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GODILO-GODLEVSKIY V.A. ET AL.: "PERSPEKTIVY PRIMENENIYA NOVYKH LABORATORNYKH METODOV V RANNEY DIAGNOSTIKE SEPSISA", MEDITSINSKIY VESTNIK MVD, vol. 75, no. 2, 2015, pages 54 - 57 *
GULIY O.I ET AL.: "Ekspress-analiz chuvstvitelnosti bakteriy k beta-laktamnym antibiotikam s pomoshchiu rezonatora s poperechnym elektricheskim polem", ANTIBIOTIKI I KHIMIOTERAPIIA, vol. 64, no. 1-2, 2019, pages 3 - 8, DOI: 10.24411/0235W2990W2019W10001 *

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