RU2766185C1 - Sample preparation method for accelerated identification of microorganisms from positive hematological cultures - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: disclosed is a sample preparation method for accelerated identification of microorganisms from positive hematological cultures, comprising collecting 5 ml of a culture medium with blood from a flask with a positive blood culture into a vacuum test tube with a separating gel, centrifuging for 12 minutes at 1,000 g, removing the supernatant without affecting the layer of cell detritus with microorganisms on the surface of the gel. 1.5 ml of sterile saline is added, the sediment is resuspended, centrifuged for 3 minutes at 100 g; 700 mcl of the supernatant is transferred into an Eppendorph test tube, centrifuged for 2 minutes at 10,000 g; the supernatant is removed, 15–25 mcl of 70 % formic acid is added to the sediment, the mixture is stirred and 15–25 mcl of acetonitrile is added, the mixture is stirred and centrifuged for 2 minutes at 10,000 g. On a target for a mass spectrometer, 1 mcl of the supernatant is applied on 2 points and a precipitate is applied on 2 additional points, dried at room temperature and coated with a matrix of α-Cyano-4-hydroxycirmamicacid; matrix drying is followed by identification on a MALDI ToF mass spectrometer.

EFFECT: invention provides identification of microorganisms without isolation of their pure culture.

1 cl, 1 tbl, 1 ex

Description

The technical field to which the invention belongs

The invention relates to medicine, namely to clinical laboratory diagnostics, medical microbiology, bacteriology, and can be used for sample preparation of positive hematological cultures for accelerated identification of microorganisms by MALDI ToF mass spectrometry.

State of the art

The problem of isolating blood cultures in case of suspected bloodstream infections is currently quite relevant. [Kargaltseva N.M., Kocherovets V.I., Mironov A.Yu., Borisova O.Yu. Method for obtaining blood culture in the diagnosis of bloodstream infection. Clinical laboratory diagnostics. 2020; 65 (3): 185-190]. At the same time, microbiological diagnosis of septic conditions consists of several stages. Thus, the modern diagnosis of bloodstream infections is based on the use of commercial nutrient media for the cultivation of microorganisms that are incubated for 2-7 days, with a positive result, the liquid nutrient medium is transferred to solid nutrient media for subsequent species identification and determination of the antibiotic sensitivity of the pathogen. Thus, the time taken from blood sampling to the issuance of the result can vary and ranges from 3 to 10 days. Of course, such a duration of the study is unacceptable, and given the high incidence of comorbid patients with sepsis, high mortality in this condition, significant economic costs in nursing patients, there is a need to accelerate the procedures for isolation, identification and determination of sensitivity to antimicrobial drugs in this group of patients.

A known method for diagnosing a bloodstream infection, in which it is proposed to take blood for seeding not from a peripheral catheter, but from a central one [Merzhvinsky I.A., Nagrodsky S.L., Basanov R.V., Feodasiadi L.A. A method for detecting bacteremia in case of suspected sepsis. No. RU 2217499 C2].

The disadvantages of this method is the duration of incubation, the complexity of the catheter, the lack of data on the amount of blood required for research.

A known method for accelerated identification of microorganisms using MALDI-ToF mass spectrometry, based on the use of the Sepsityper technique, the essence of which is to take 1.0 ml of the contents of a vial from a positive blood culture, subsequent purification procedures, which include the use of a number of reagents for and centrifugation series in different acceleration modes.

The disadvantages of the method are high labor intensity, the need to use additional expensive reagents [Polishchuk A.G. MALDI-TOF Mass Spectrometric Identification of Medically Significant Micromycetes (Review). Problems of Medical Mycology, 2011, Vol. 13, No. 4].

A known method for diagnosing bacteremia, based on taking 4.5 ml of whole blood of a patient with fever, centrifugation, isolation of leukocyte suspension and subsequent inoculation of leukocyte suspension on blood agar [Kargaltseva N.M. Method for diagnosing bacteremia. No. RU 2098486 C1]. A significant disadvantage of this method is the limitation of the use of this invention in patients with leukopenia, cancer patients, patients with hemocytopenia.

A known method for accelerated identification of microorganisms based on the study of positive blood culture samples [Amineva P.G., Rudnov V.A., Karmatskikh O.G., Nevskaya N.N., Velsky D.V., Ivanova N.A. Results of identification of bacteria from positive blood cultures of patients of a multidisciplinary hospital using MALDI-TOF mass spectrometry. Clinical microbiology and antimicrobial chemotherapy. 2018 V. 20 No. 4]. This method is taken as a prototype.

The disadvantage of this method is a technique that involves the use of saponins and centrifugation at high acceleration values, which is associated with excessive sedimentation of bacteria along with blood cells and cell debris, which can reduce the identification efficiency.

Disclosure of invention

The aim of the invention is to develop a method for the accelerated identification of the causative agent of bloodstream infections from a positive sample of hematological culture, without the steps of isolating the microorganism on dense nutrient media for the etiological diagnosis of diseases accompanied by bacteremia or sepsis.

This is achieved by the fact that, in contrast to the known methods of sample preparation for accelerated identification of microorganisms from positive hematological cultures, 5 ml of a nutrient medium with blood are transferred from a vial with a positive blood culture into a vacuum tube with a separating gel, then the tube is centrifuged for 12 minutes at 1000 g , the supernatant is drained from the tube without affecting the layer of cellular detritus with microorganisms, which remains on the surface of the gel. Then 1.5 ml of sterile saline is added and the pellet is resuspended. Next, the material is centrifuged at 100 g for 3 minutes, after which 700 μl of the supernatant is taken, which is transferred to an Eppendorf-type tube, then the suspension is centrifuged at 10,000 g for 2 minutes. The supernatant is removed, 15-25 µl of 70% formic acid is added to the precipitate obtained, followed by mixing, then 15-25 µl of acetonitrile is added, followed by mixing, centrifuged again at 10,000 g for 2 minutes. Next, 1 µl of supernatant for 2 points is applied to the target for the mass spectrometer, then the supernatant is removed, a precipitate is applied with a disposable microbiological loop for 2 additional points, dried at room temperature and covered with a SNCA matrix (α-Cyano-4-hydroxycirmamicacid), left at room temperature. temperature until completely dry; followed by identification on a MALDI ToF mass spectrometer.

The technical result of the invention lies in the fact that the claimed method of sample preparation of positive hematological cultures for accelerated identification of microorganisms by MALDI ToF mass spectrometry allows obtaining a sufficient amount of the biological mass of microorganisms purified from the components of the nutrient medium and blood.

Implementation of the invention

The essence of the proposed method is as follows: 5 ml of a nutrient medium with blood is transferred from a vial with a positive blood culture into a vacuum tube with a separating gel, then the tube is centrifuged for 12 minutes at 1000 g, the supernatant is drained from the tube without affecting the layer of cellular detritus with microorganisms, which remains on the surface of the gel. Then 1.5 ml of sterile saline is added and the pellet is resuspended. Next, the material is centrifuged at 100 g for 3 minutes, after which 700 μl of the supernatant is taken, which is transferred to an Eppendorf-type tube, then the suspension is centrifuged at 10,000 g for 2 minutes. The supernatant is removed, 15-25 µl of 70% formic acid is added to the precipitate obtained, followed by mixing, then 15-25 µl of acetonitrile is added, followed by mixing, centrifuged again at 10,000 g for 2 minutes. Next, 1 µl of supernatant for 2 points is applied to the target for the mass spectrometer, then the supernatant is removed, a precipitate is applied with a disposable microbiological loop for 2 additional points, dried at room temperature and covered with a SNCA matrix (α-Cyano-4-hydroxycirmamicacid), left at room temperature. temperature until completely dry; followed by identification on a MALDI ToF mass spectrometer.

The proposed method allows the identification of microorganisms grown in positive hematological cultures without the need to isolate their pure culture.

The proposed method is characterized by simplicity, no need to use special reagents (formic acid, acetonitrile, hydroxycinnamic acid are reagents used in the standard procedure for identifying microorganisms by MALDI-ToF mass spectrometry), equipment, reliability and efficiency.

The implementation of the proposed method can be demonstrated by the following examples:

Example 1

Patient K., 56 years old, was admitted to the hospital for a planned course of polychemotherapy for a systemic tumor blood disease. After the course, the patient against the background of agranulocytosis began to experience high fever, chills, sweating, tachycardia. To exclude sepsis, blood cultures were carried out in vials for the cultivation of aerobic and anaerobic microorganisms. Growth was registered in an aerobic flask after 26 hours 34 minutes, after which 200-400 μl of the sample from the positive flask were inoculated onto solid nutrient media to isolate a pure culture of the pathogen; in parallel with this, a study of a positive sample of blood culture was carried out by the proposed method. The result of the identification of the microorganism by the proposed method - Staphylococcus aureus. The growth of colonies on dense nutrient media was registered after 19 hours, identification by MALDI-ToF mass spectrometry, the result was confirmed - S. aureus was isolated.

Example 2

Patient C was admitted to the clinic with a clinical picture of femoral phlegmon on the left. History of cadaveric kidney allotransplantation for chronic kidney disease, immunosuppressive therapy. An autopsy was performed, phlegmon drainage was performed, broad-spectrum antibiotics were prescribed to prevent postoperative complications. Within a few days, there was a positive trend, but on the 4th day the patient's temperature rose to 38-39°C, fever, chills, tachycardia were noted. Laboratory revealed leukocytosis with toxic granularity, an increase in the content of C-reactive protein. To exclude sepsis, blood cultures were carried out in vials for the cultivation of aerobic and anaerobic microorganisms. Growth was recorded in an aerobic flask after 16 hours, after which 200-400 μl of the sample from the positive flask were inoculated onto solid nutrient media to isolate a pure culture of the pathogen; in parallel with this, a blood culture study was carried out by the proposed method. The result of the identification of the microorganism by the proposed method - Klebsiella pneumonia. The growth of colonies on dense nutrient media was registered after 15 hours, identification by MALDI-ToFMacc-spectrometry, the result was confirmed - Klebsiella pneumonia was isolated.

Thus, a survey was conducted in 49 patients of therapeutic and surgical profiles, in the diagnosis of sepsis. The coincidence of identification results occurred in 94% of cases. The identification results are presented in Table 1.

Claims (1)

A method of sample preparation for accelerated identification of microorganisms from positive hematological cultures, characterized in that 5 ml of a nutrient medium with blood is transferred from a vial with a positive blood culture into a vacuum tube with separating gel, then the tube is centrifuged for 12 minutes at 1000 g, the supernatant is drained from the tube, without affecting the layer of cellular debris with microorganisms that remains on the surface of the gel; then 1.5 ml of sterile saline is added, the precipitate is resuspended, then the material is centrifuged at 100 g for 3 minutes, after that 700 μl of the supernatant is taken, which is transferred into an Eppendorf-type tube, then the suspension is centrifuged at 10,000 g for 2 minutes ; the supernatant is removed, 15-25 μl of 70% formic acid is added to the resulting precipitate, followed by stirring, then 15-25 μl of acetonitrile is added, followed by stirring, centrifuged again at 10,000 g for 2 min, then applied to the target for the mass spectrometer 1 µl of supernatant per 2 points, then the supernatant is removed, sediment is applied to 2 additional points with a disposable microbiological loop, dried at room temperature and coated with CHCA (α-Cyano-4-hydroxycirmamicacid) matrix; further, after complete drying of the matrix, identification is carried out on a MALDI ToF mass spectrometer.