RU2616249C1 - Method for express-diagnostics of blood flow infection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to laboratory methods of research, express-method for diagnostics of infectious disease accompanied by presence of microorganisms in blood. Way of express-method of blood flow infection diagnostics includes identification of microorganisms in blood by method of blood smear microscopy with immersion and magnification ×1000, with smear being prepared from leukocyte layer of venous blood with application of "two glasses" technique. After drying distilled water is poured on smear, covering its entire area, after 3-5 s destructed erythrocytes are washed away and smear is dried. After fixation above burner flame and Gram-staining, microscopy of obtained smear is realised by provision of colourless background of blood smear.

EFFECT: invention ensures increased efficiency of diagnostics of blood flow infection due to possibility of identification of Gram-positive and Gram-negative microorganisms with preservation of simplicity and availability of diagnostics.

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Description

The invention relates to medicine, namely to laboratory research methods, an express method for the diagnosis of an infectious disease, accompanied by the presence of microorganisms - its pathogens - in the blood.

Microscopic examination of biological material (urine, feces, sputum, separated by purulent-inflammatory foci) is an obligatory step in the clinical study of material in a clinical laboratory.

The microbiological laboratory also conducts a microscopic examination of biological material (sputum, cerebrospinal fluid, wounds, punctures, etc.) for certain infections, but microscopy of a peripheral blood smear has not been widely used in the diagnosis of infection in the blood, despite the fact that used since 1906, when meningococcemia was diagnosed by detecting meningococci in a smear of peripheral blood.

The method of microscopy of biological material, including blood, refers to accelerated methods for detecting a microorganism, since the clinician has information 1-1.5 hours after the material is received in the laboratory. In stained smears, bacteria and fungi are detected on the basis of morphological and tinctorial characters. In the future, blood microscopy began to be used to diagnose life-threatening infectious diseases, since it allowed to detect the pathogen within 1-1.5 hours.

Historically, the diagnoses of many diseases were made based on the results of microscopy.

The first report of the detection of bacteria in peripheral blood by a microscopic method was made by Raver and Davaine in 1850. They saw little bodies in the blood of a sheep that were artificially introduced from a sheep who died of anthrax. In 1906, F.W. Andrews found meningococci in a peripheral blood smear before receiving a blood culture [1].

In 1915, Coze and Feltz reported the presence of bacteria in the blood of patients suffering from typhoid fever [2]. In 1918, W.W. King examined a drop of blood taken from the earlobe in patients with cerebrospinal meningitis and found gram-positive and gram-negative cocci, which were subsequently isolated and identified as Staphylococcus aureus and Neisseria meningitidis [3].

The microscopic method of blood testing has been widely used in the diagnosis of parasitic diseases that had a high mortality rate. Babesia microti was first diagnosed in 1888 as a cause of death for cattle and dogs. Anderson et al. published a case of babesiosis in humans after a tick bite. Microscopy of the blood revealed parasites in red blood cells in the form of bacteria, dividing into two or more daughter cells [4]. The studied material was a drop of blood.

The technique for preparing a blood smear has undergone changes.

Initially, a “thick drop" preparation was prepared for microscopic examination of blood [5]. Thus, the diagnosis of malaria was introduced on the basis of microscopy of a thick drop of blood, which is widely used to this day.

Since the introduction of thin blood smear microscopy into clinical laboratory practice using the “two glass” technique, the era of diagnosing a wider range of microorganisms, including pathogens of life-threatening diseases (meningococcus), has begun. Improving the preparation of a blood smear contributed to the diagnostic expansion of the spectrum of pathogens, including bacteria and fungi.

In 1944, A. Humphrey developed a technique for producing a white blood cell concentrate from a peripheral blood sample and described a technique for preparing a smear of peripheral blood from a given blood layer. Smears were prepared using the “two glass” technique [6, 7, 8]. The author was able to diagnose bacteremia before receiving a blood culture. The advantage of this method was that the elements of the blood under a certain centrifugation mode were distributed in accordance with their gravity. Heavy red blood cells settled to the bottom, light plasma was at the top, and under the plasma on the red blood cells lay a thin white layer, which is a mixture of white blood cells, platelets and microorganisms glued to them. Some microorganisms were located inside neutrophils due to their phagocytosis, and some microorganisms were glued together by platelets. Extracellular microorganisms were raised by platelets through the medium to the leukocyte layer. Staphylococci and meningococci were located extra- and intracellularly in blood smears. The option of staining blood smears according to Wright and Gram made it possible to quickly detect staphylococci, streptococci and meningococci in patients with septicemia.

Blood smears were thin, the cellular elements of the blood were located in one layer and were clearly visible. When viewed under a light microscope, gram-positive microorganisms were well detected and gram-negative, with great difficulty, were found, since they were the same color as red blood cells forming the background of the smear.

Comparison of two methods (microscopy and culture) in the diagnosis of bacteremia and fungemia showed the advantage of microscopy over culture. This is because the cultural method of isolating the pathogen from the blood requires the use of highly nutritious media (cardio-cerebral), anaerobic cultivation conditions, closed blood culture systems and a certain amount of time for the growth of the microorganism. Unfortunately, in Russia they do not produce in an industrial way cardio-cerebral media for the cultivation of blood, anaerobic cultivation conditions are used only in laboratories of large medical centers. Currently, for the purpose of diagnosing bacteremia, foreign expensive closed systems are used only by individual large medical institutions. For the selection of mushrooms, separate special media are required.

The microscopic method of blood testing allows you to detect bacteria and fungi, diagnose bacteremia and fungemia in a short period of time. Based on the morphology and tinctorial properties of the detected microorganisms, it is possible to prescribe empirical antimicrobial therapy within 1-1.5 hours from the moment of blood entering the laboratory.

The microscopy method is used to diagnose bacterial and fungal infections.

The fulminant form of meningococcemia was diagnosed by blood smear microscopy to detect gram-negative diplococci in polymorphonuclear cells. In addition to the phagocytized ones, there were extracellular diplococci [26]. Earlier, in 1935, Boone and Hall [27] and in 1943 Thomas [28] diagnosed cases of meningococcemia based on microscopic findings from a peripheral blood smear.

Microscopy of smears of the leukocyte layer of a peripheral blood sample was used to diagnose disseminated infection caused by Mycobacterium avium complex in a patient with immunodeficiency syndrome. All patients later received blood cultures of this pathogen. The sensitivity of the microscopic method was 35%, the specificity and degree of assumption of these pathogens was 100%. The authors showed that smear microscopy of the leukocyte layer in case of mycobacterial infection is a quick, simple and specific method for diagnosing disseminated infection [29].

Acid-resistant bacteria can be detected in blood smears by circulating them in the bloodstream in an amount of 20-180 CFU / ml of blood in patients without appropriate therapy and from 800 CFU / ml of blood or more in patients during therapy [29].

Graham et al. found acid-resistant bacteria in smears of the leukocyte layer when stained by the Kignon method in the presence of bacteria in the bloodstream in the amount of 150,000 CFU / ml of blood in patients with AIDS and disseminated infection [30]. The authors claimed that the sensitivity of the smear microscopy method of a white blood cell layer depends on the amount of mycobacteria in the blood. Mycobacteremia progressively increased in patients with AIDS and diagnosis was possible with a microscopic examination of the blood.

Microscopic examination of a blood smear made it possible to determine the disseminated form of toxoplasmosis [31].

Pneumococcal bacteremia was diagnosed by microscopy of a peripheral blood smear of a patient with myeloma. A blood smear was stained by Wright and diplococci were located extracellularly. In blood culture and cerebrospinal fluid culture pneumococcus was obtained [32].

Microscopy of blood smears of a patient with septicemia against the background of chondrosarcoma revealed spores of Cl. perfringens inside white blood cells. Smears were made from a white blood cell layer and stained with Wright. The obtained hemocultures of the spore-forming microorganism confirmed the previously detected pathogen in smears [33].

The diagnosis of systemic candidiasis was made by microscopy of “thin” smears and smears of the leukocyte layer of a peripheral blood sample.

In 1945, Parsons and Zarafonetis found Histoplasma capsulatum blastospores inside neutrophils in a “thin” smear of peripheral blood [34]. Other authors described fungemia caused by Histoplasma capsulatum, which was diagnosed microscopically before receiving a blood culture. The smear was made of a white blood cell layer and stained according to Wright-Giemsa. In the smears, small contrasting bodies that were extracellular were visible. A blood culture was later obtained [35].

In 1948, Portnoy described two cases of detection of C. albicans in “thin” smears of peripheral blood before receiving a blood culture [36]. Anderson in 1972 described the detection of yeast cells in brands of peripheral blood in systemic candidiasis. The diagnosis was made on the fact of the presence of yeast cells and pseudohyphus in a blood smear [37]. In 1973, Silverman diagnosed systemic candidiasis by microscopy of smears of the white blood cell layer [38]. Most of the detected microorganisms were blastospores in the form of small basophilic oval bodies surrounded by a clear zone. Most of them were in the cytoplasm of neutrophils. Smears were stained according to Wright and methenamine silver [38]. When stained with PAS (Periodic Acid-Schiff), the yeast cells and pseudohyphae are stained in bright red. The author suggested using the method of microscopy of the white blood cell layer to confirm the expected candidaemia [38]. K. Kobza demonstrated the detection of blastospores and pseudohyphus in the free and phagocytosed by leukocyte states in smears of the leukocyte layer of a peripheral blood sample and the growth of S. aureus and C. albicans in blood culture, which confirmed microorganisms previously found in blood smears [39].

Fungal elements, including blastospores, pseudohyphae were found in the patient after surgery. Yeast cells were located both intracellularly and outside the cells. Two days after the microscopic diagnosis, C. albicans blood culture was obtained, which confirmed the initial microscopic findings [40].

In 1986, the first case of diagnosing Candida parapsilosis with a microscopic blood test was published. Yeast cells were located inside monocytes and neutrophils. The patient suffered from chronic lymphatic leukemia [41]. Microscopy of peripheral blood smears in neutrophils revealed phagocytosis of the cells of the fungi Rhodotorula rubra and Candida guilliermondii in patients with neutropenia.

Red blood cell hemolysis occurs when the red blood cell membrane is destroyed. Destruction can occur in the bloodstream and outside the body, i.e. in vitro. When a red blood cell enters the water, it begins to absorb it. Water, due to the sharp difference in the osmotic pressure inside and outside the red blood cell, quickly diffuses into the red blood cell, causing it to swell and rupture of the membrane. Since the osmotic pressure of water is less than the pressure inside the red blood cell, therefore, the water enters the red blood cell, increasing its volume and pressure inside. The red blood cell swells, and its membrane breaks and coagulates [42]. In experiments, the rate of hemolysis was shown. Every 30 seconds, 30% of red blood cells undergo hemolysis. In the patent "Method for determining the osmotic resistance of red blood cells", the degree of hemolysis of red blood cells in various solutions was evaluated. It turned out that in distilled water hemolysis occurs in 100% of cases [42].

As a prototype of the invention, a method for preparing a blood smear from a leukocyte layer of venous blood is described in the methodological recommendations “Microbiological methods for the diagnosis of blood flow infection”, 2010, approved by the Health Committee of the Government of St. Petersburg [43]. The above prototype of the invention provides for the preparation of a smear, which is as follows.

Blood is taken into a test tube containing an anticoagulant, after centrifugation, the upper light layer, the plasma, is removed and a thin white layer lying on the red blood cells, which is a leukocyte layer, is collected. Drops of this layer are applied to slides and using a “two glass” technique, a thin smear is made with a grinding glass. The smear is dried, fixed, stained according to Gram and viewed under a light microscope with immersion. A smear consists of red blood cells arranged in a single layer, creating a red background, and white blood cells located between them with blue-stained nuclei and microorganisms of different shapes and color of staining (red and blue). Under microscopy, gram-positive microorganisms of blue are clearly visible on a red background, and gram-negative red are merged with the background and are difficult to see. Therefore, in the etiological diagnosis of bloodstream infection, in most cases, gram-positive microorganisms were detected, including bacteria and fungi.

We set the task to develop an affordable universal method for the express method for the diagnosis of blood flow infection, which allows to detect both gram-positive and gram-negative microorganisms in the patient's blood.

The technical result achieved by the implementation of the invention is to increase the efficiency of the diagnosis of blood flow infection due to the additional ability to identify gram-negative microorganisms by providing a colorless blood smear background while maintaining simplicity and accessibility of diagnosis.

We proposed to improve the diagnostic capabilities of a method for detecting bloodstream infection to eliminate the pink background of a blood smear by destroying red blood cells. Thus, processing a blood smear with distilled water destroys and removes red blood cells from a blood smear, which provides additional detection of gram-negative (red) microorganisms against a colorless blood smear background under microscopy.

The proposed method allows guaranteed detection of all available microorganisms in a blood smear, both gram-positive and gram-negative, since all colored forms of bacteria and fungi lie on a transparent background of a blood smear.

Thus, the sensitivity of the method increases due to the detection of gram-negative microorganisms. If gram-positive microorganisms make up about 50-60% in the etiology of bacteremia, then gram-negative microorganisms account for about 20%, therefore, the detection of gram-negative microorganisms can increase the diagnostic efficiency by 20%, thereby increasing the value of the method and the possibility of selecting antimicrobial therapy.

In addition, the detection of gram-negative microorganisms is important for the diagnosis of hospital infection and secondary infectious endocarditis (after surgery for prosthetic heart valves). Gram-positive microorganisms predominate in the etiology of bacteremia in outpatients, and gram-negative in hospital infections. Deciphering the etiology of hospital infections is the key to preventing hospital infection in each particular clinic.

When comparing the percentage of detection of microorganisms in the blood and the percentage of their excretion during blood culture, it turned out that the microscopic method is several times more effective than the cultural method. The percentage difference indicates that even the richest nutrient medium (cardiac) with hemolyzed blood and vitamin supplements cannot recreate the complete set of essential nutrients for the vital functions of microorganisms that the blood possesses. In addition, with microscopy, both live and dead cells of microorganisms can be detected, and when blood is sown, we get the growth of only living microorganisms.

Empirical antimicrobial therapy is prescribed for detected microorganisms in a blood smear. From the time of preparation of the smear to the appointment of therapy requires 1-1.5 hours. This method of diagnosing an infection in the blood refers to the express method.

The invention consists in the following.

The method of the express method for the diagnosis of blood flow infection involves the identification of microorganisms in the blood by a blood smear microscopy with immersion and magnification × 1000. In this case, a smear is prepared from a leukocyte layer of venous blood using the “two glass” technique. After drying, distilled water is poured onto the smear, covering its entire area. After 3-5 seconds, the destroyed red blood cells are washed off and the smear is dried. After fixing over the flame of the burner and staining according to Gram, microscopy of the resulting mask is performed.

The method is as follows.

The patient’s blood is collected in a “syringe tube” with an anticoagulant (citric acid sodium) in a volume of 5 ml or 10 ml by venipuncture. After removing the syringe from the vein, the needle is removed, the piston is broken off and the resulting tube from the syringe is turned over at least 3 times for good mixing of blood with the anticoagulant. After delivery to the laboratory, the blood tube is centrifuged at 1000-1500 rpm for 15-20 minutes. The Pasteur pipette removes the plasma. The leukocyte layer lies on red blood cells in the form of a thin light layer with a height of 0.5-1.0 mm. A capillary of Panchenkov or a disposable pipette with a wide flat end collect this layer and drop it onto a glass slide. Immerse the grinding end of the slide in a drop, wait for the drop to spread over the edge of the grinding glass and, gently pressing the smear glass, distribute the drop of the leukocyte layer on the surface of the slide with the “two glasses” technique to obtain a thin smear of blood. The "two glass" technique is carried out in clinical laboratories for the preparation of thin clinical blood smears. A blood smear is dried in air. The smear is pink in color. Then distilled water is poured onto the smear and the red blood cells destroyed within a few seconds are washed off. The smear takes the form of transparent glass. The smear is dried again. Then it is fixed above the flame of the burner and stained according to Gram according to the generally accepted technique. A blood smear is viewed under immersion oil with a magnification of × 1000. Microscopy of a smear treated with distilled water on a white background clearly shows the morphological forms of microorganisms stained both gram-positive and gram-negative. Microorganisms, painted gram-negative in a slightly pink color, are clearly visible on a transparent glass background.

To prove the possibility of realizing the claimed purpose and achieving the specified technical result, we provide the following data.

Example 1

Blood smears were studied, prepared from the leukocyte layer of a peripheral blood sample, by the microscopic method of patient G., 37 years old, with a clinical diagnosis of infectious endocarditis. Two blood smears were prepared for Gram staining with preliminary treatment with distilled water (according to the proposed method) and without treatment. Microscopy of a blood smear without treatment with distilled water revealed gram-positive yeast cells of blue color on a dense bright pink background of red blood cells with a small number of white blood cells. Microscopy of a blood smear with pretreatment with distilled water revealed pink gram-negative rods, blue gram-positive yeast cells, and white blood cells with blue-stained nuclei inside them in small quantities. All forms were clearly visible on a white transparent glass background. During blood culture, an E. coli blood culture was obtained, which confirmed the presence of E. coli in the smear, but seen only on a transparent background, i.e. without red blood cells.

Example 2

In the same way, smears of patient I., 50 years old, were prepared with a diagnosis of chronic inflammatory disease of the upper respiratory tract. Microscopy of the smear without treatment with distilled water revealed gram-positive cocci in a small amount and white blood cells in a significant amount. In a blood smear, which was subjected to treatment with distilled water (according to the proposed method), on a transparent background, gram-negative bacilli and gram-positive cocci in a small amount, a large number of leukocytes were visible. In blood culture, a polymicrobial blood culture was obtained containing strains of Staphylococcus epidermidis and Enterobacter aerogenes.

The table shows the results of the detection of gram-negative rods under microscopy of a blood smear under different conditions for the preparation of a blood smear (results of parallel microscopy of blood smears).

The proposed method is technically simple in terms of the method and reagent used, effective for improving the detection of microorganisms, and is available for the microbiological laboratory for microbiological blood tests for infections in the blood.

Literature

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Claims (1)

A method for the rapid diagnosis of blood flow infection, including the detection of microorganisms in the blood by microscopy of a blood smear with immersion and magnification × 1000, characterized in that the smear is prepared from a white blood cell layer using the “two glasses” technique, after drying, distilled water is poured onto the smear, coating its entire area, after 3-5 seconds, the destroyed red blood cells are washed off, the smear is dried, then after fixation over the flame of the burner and staining according to Gram microscopy of the obtained smear is performed.