RU2144190C1 - Method of diagnosis of staphylococcus infection - Google Patents

Abstract

FIELD: medicine and microbiology; applicable in making of diagnosis and identification of pathogen of staphylococcus infection. SUBSTANCE: method is based on blood analysis. Method includes introduction of alive day culture of staphylococcus into preliminarily separated lymphocytes; incubation for 3 h. After coloring, presence of staphylococcus infection and degree of sensitization are determined by per cent of cells joining to their membrane of five and more cells of microorganisms. The method provides for reduced time of diagnosis by 87.7%. EFFECT: higher sensitivity, prevention of errors in making diagnosis, provision of clinico-immunological control for quality of medical manipulations, prevention of recurrences, saving of labor input of medical staff, possibility of early starting of treatment. 2 tbl, 2 ex

Description

 The invention relates to medicine and microbiology and can be used in all areas related to medicine in establishing the diagnosis and identification of the pathogen.

 Over the past decades, the diagnosis and treatment of diseases associated with staphylococcal infection has remained an unresolved practical public health problem. This is due to the significant spread of the pathogen in the environment (94.4 - 97.3% of children upon discharge from the hospital are carriers of pathogenic staphylococci), pronounced resistance of staphylococci to adverse factors, difficulty in diagnosis, a wide range of clinical manifestations - from erased asymptomatic to severe generalized forms, a tendency to a protracted and chronic course, a decrease in sensitivity to antibiotics due to the appearance of antibiotic-resistant strains of staphylococci, a change biological properties, increased their pathogenicity and virulence. Staphylococcal infection takes first place in the etiology of acute and chronic forms of diseases such as sinusitis, otitis media, pneumonia, tonsillitis, bronchitis, infectious allergic bronchial asthma, meningitis, sepsis, pyelonephritis, osteomyelitis, purulent skin and subcutaneous tissue lesions. Moreover, having pronounced antigenic activity, staphylococcus sensitizes the body, being a "trigger factor" for the occurrence of eczema, neurodermatitis, psoriasis. So, in the pathogenesis of true eczema, the role of sensitization to staphylococcal allergens coming from foci of focal infection is not in doubt. To detect allergies to pyococci, numerous allergological and immunological diagnostic methods are used both in vitro and in vivo. The abundance of diagnostic methods indicates that none of them has independent significance and should be used in combination with other methods and taking into account the clinical picture and medical history.

 A known method for the diagnosis of staphylococcal infection using the reaction of passive (indirect) hemagglutination (RPHA). (Dolmatov V.V. Diagnosis of staphylococcal diseases in children and adults: Method of recommendation. - M., 1989, - 3-10 pp.). It is based on a method of indirect Boyden’s indirect hemagglutination. The essence of the reaction is that standard staphylococcal L-toxin is adsorbed on red blood cells and red blood cells agglutinate in the presence of appropriate serum. Staphylococcus antigens obtained by ultrasonic disintegration, native staphylococcal toxoid can serve as an antigen in RPHA.

 The method has a low sensitivity (the reaction is positive in 6-40% of cases in patients with staphyloderma; in 50% with gastroenterocolitis, in 82.5% with sepsis); uninformative in identifying the severity and severity of the disease, does not give an idea of the sensitization of the body by staphylococci.

For the prototype, we adopted a method for the diagnosis of staphylococcal infection (V. Silich. A protracted, recurring and chronic course of staphylococcal infection and its diagnosis. - Kiev .: Health, 1991. - 35-39 p.), Including the detection of antibodies to toxins in serum staphylococcus. To set up the reaction, 0.5 ml of the patient’s blood serum and standard staphylococcus L-toxin (CAAT) are taken in an amount of 0.1 ml. Take 0.3 ml of suspension in another tube. Incubate the first tube for 1 hour at 37 ^o C in a thermostat, and the second at 4 ^o C in the refrigerator. As control using standard serum. Then all tubes are photometric; bacteriostatic activity of blood serum (BASK) is calculated by the formula. BASK characterizes the body's ability to resist staphylococcal infection.

The disadvantages of the method, in addition to those noted in the analogue, include
- the need for repeated blood tests in the same patient to establish a diagnosis due to the extremely wide range of individual norms;
- the impossibility of use in children: even in the presence of staphylococcal infection, the content of CAAT remains within normal limits, because the body of children has little ability to produce these antibodies;
- a clear dependence on the form of the disease: with tonsillitis and rhinosunusitis, the level of CAAT is increased, and with purulent otitis media, the titers of CAAT remain within normal limits, despite the repeated isolation of Staphylococcus aureus from the separated middle ear (with otitis media, staphylococci form L-toxin to a lesser extent, producing more other toxins (leukocidin);
- lack of correlation with the period of the disease. In the acute process, a low content of CAAT (not diagnostically significant) is detected, which increases by the time of recovery. In chronic diseases, due to the depletion of the immune response, CAAT is also small. Thus, the method is characterized by reliability and is unreliable in the diagnosis.

 Objectives of the invention: the development of a gentle express method for the diagnosis of staphylococcal infection in children and adults, with sensitivity, reliability, the ability to correlate with the severity of the disease, informative regarding the sensitization of the patient's body.

 The essence of the invention is an in vitro blood test, preliminary isolation of lymphocytes, the introduction of a live daily culture of staphylococcus into the sample, incubation for 3 hours and after staining by the percentage of cells that have attached 5 or more cells of microorganisms to their membrane, determining the presence of staphylococcal infection and the degree sensitization.

 The method is as follows.

Take the blood from the patient’s ulnar vein to an in vitro tube with Trilon B (5 μg Na 2 EDTA per 5 ml of blood. Mix the blood with dry powder of Na 2 EDTA thoroughly by gently swaying the tube from side to side for 30-60 sec. to obtain a homogeneous (homogeneous) suspension. This blood is layered on 1.5-2 ml of a mixture of ficoll-verographin (d - 1,077 g / ml). Centrifuged this mixture for 35-40 minutes at 1500 rpm, a ring of lymphocytes is isolated and transferred in a Florinsky test tube, lymphocytes are washed twice with medium 199 or buffered saline. itov resuspended in 1 ml of 199 medium or buffered saline, prepared a working concentration of the main ingredients of the reaction (0.5 billion. aureus and microbial bodies 2 • ¹⁰ June lymphocytes in 1 ml). To 0.1 ml of lymphocyte poured 0.1 ml of a live culture of staphylococcus at a working concentration Incubate the mixture in a thermostat at 37 ^o C for 150 minutes Continue incubation at room temperature for another 30 minutes Precipitate the resulting complexes by centrifugation at 1000 rpm for 5 minutes Rosettes are fixed by adding 0.05 ml of a 0.6% glutaraldehyde solution for 15 minutes. Fixation is stopped after adding 0.5 ml of distilled water. Centrifuge the suspension for 5 minutes at 1000 rpm. The supernatant is drained, 0.1 ml of saline solution is added to the precipitate and pipetted carefully. Transfer the contents of the test tube to fat-free glass, make a smear, dry it at room temperature, fix it with methyl alcohol and stain with a 0.5% safranin solution in a 0.25% borax solution, and the core is stained with a 1% brilliant green solution. The results of the reaction of rosette formation are taken into account under immersion, 200 cells (lymphocytes) are counted, and% of cells that attach 5 or more microorganism cells to their membrane that are considered rosette are calculated.

 The empirical method revealed that the degree of sensitization is determined under a microscope by counting% of cells that have attached 5 or more cells of microorganisms to their membrane, while in case of% of attached cells it was 15.28 ± 1.01% for S. aureus, for S epidermidis 26.56 ± 1.06, normal state was determined. With an increase in this level, pathology is revealed for S. aureus and S. epidermidis. Moreover, the higher this level, the higher the sensitization, and the infection is more stable.

 The whole procedure for performing this diagnosis takes 3.5 hours in time (tab. 1, 2).

 Conclusion: from table 1 it is seen that the proposed method has an advantage in all of the above parameters and is free from the disadvantages of the prototype.

 Conclusion: from table 2 it is seen that in patients suffering from staphylococcal diseases, the level of sensitization to staphylococci increases, and a correlation between the degree of sensitization and the severity of the disease is clinically observed.

 The method was tested under stationary conditions in the regional dermatovenerological dispensary of Krasnodar.

Example 1. Patient T., 22 years old, was hospitalized in February 1997 in the Krasnodar Regional Dermatovenerologic Dispensary with a diagnosis of true eczema in the acute stage. Concomitant disease: chronic tonsillitis. Upon admission, the patient complained of weeping and itching in the upper limbs for 2 years. For the first time, eczema manifested itself after strong feelings while serving in the Army. Angina has been sick since childhood 2-3 times a year. He was repeatedly treated by a dermatologist at the place of residence, but relapses continued 2-3 times a year. She is registered with an ENT doctor. On the day of admission, blood was taken in vitro for a specific rosette reaction: a suspension of lymphocytes isolated on a ficoll-verographin (0.1 ml) isolated on a gradient was combined with an equal volume of daily staphylococcus culture. Incubated for 2 hours 30 minutes at 37 ^° C and 30 minutes at room temperature, precipitated by centrifugation for 5 minutes at 200 g. The resulting rosettes were fixed with 0.05 ml of a 0.6% glutaraldehyde solution for 15 minutes at room temperature. Fixation was stopped by adding 0.5 ml of distilled water to the tube, centrifuged for 5 minutes, the supernatant was drained. 0.1 ml of a 0.85 NaCl solution was added to the precipitate and carefully resuspended with a Pasteur pipette. A preparation was prepared on a defatted glass slide, which, after drying, was stained with a 0.5% safranin solution, followed by coloring with a 1% alcohol solution of brilliant green. The percentage of lymphocytes adhering to staphylococci was determined: the average degree of sensitization for S. aureus was 38.28 + 1.01% (644.9 + 32.21), for S. epidermidis it was 40.59 + 1.06 (1200 , 19 + 49.87). On the twelfth day of treatment, taking into account the effect on the focus of focal infection and staphylococcal sensitization (abactal and staphyloprotectin), the skin process completely regressed. The results of repeated examinations demonstrated the normalization of most immunological parameters. The reaction of specific rosetting with S. aureus - 15.29 + 1.02 (344.9 + 32.21), with S. epidermidis - 26.58 + 1.06 (600.19 + 49.87).

 Conclusion: so. the patient’s recovery was accompanied by a decrease in the number of rosette-forming cells and the normalization of sensitization.

Example 2. Patient A., 34 years old, was hospitalized in November 1997 in the CCVC with a diagnosis of true eczema in the acute stage. From concomitant diseases: recurrent suppurative otitis media on the right. Upon admission, the patient complained of weeping and itching in the upper and lower extremities. More than 7 years of relapse 1-2 times a year. The onset of the disease does not associate with anything. Otitis exacerbates 3-4 times a year for 4 years. More than once I turned to the otolaryngologist, who prescribed traditional treatment. On the day of admission, the patient received in vitro venous blood for the number of antigen-dependent rosette cells. A live daily culture of staphylococci was combined with an equal amount (0.1 ml) of lymphocytes. They were incubated at 37 ^° C for 2 hours and 30 minutes at room temperature, the rosettes were precipitated by centrifugation and fixed with a 0.6% glutaraldehyde solution. The drug was transferred to glass and stained with a 0.5% solution of safranin and 1% solution of brilliant green. Counting outlets produced under immersion. A high level of sensitization was detected with S. aureus - 45.61 + 7.15%, with S. epidermidis - 51.77 + 9.13. Prescribed treatment, including the correction of microbial allergies. On the eighteenth day of hospitalization, there were only hyperpigmented spots on the site of the former foci. Control blood tests were performed: the reaction of a specific rosette with Staphylococcus aureus - 20.17 + 1.04%, with epidermal staphylococcus -31.22 + 1.07%.

 Conclusion: the normalization of the number of rosette-forming cells was accompanied by clinical recovery.

 Thus, the proposed diagnostic method provides a reduction in the time of diagnosis by 87.7%, allowing you to start treatment earlier, and saves the hours of medical staff. The method has a high sensitivity, prevents diagnostic errors; makes it possible to carry out clinical and immunological control over the quality of the performed manipulations, prevents the occurrence of possible relapses, and therefore, significantly increases the therapeutic and economic effect.

 The developed diagnostic method is simple and affordable for widespread use in a hospital and outpatient setting.

Claims (1)

 A method for diagnosing a staph infection, including an in vitro blood test, characterized in that lymphocytes are preliminarily isolated from the blood, a live daily culture of staphylococcus is introduced into the sample, incubated for 3 hours, and after staining, according to the percentage of cells that have attached 5 or more microorganism cells to their membrane , determine the presence of staphylococcal infection and the degree of sensitization.

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