SU1534396A1 - Method of differential diagnosis of bacterial and virus meningitus - Google Patents

Abstract

The invention relates to medicine, in particular to medical microbiology, and can be used in the diagnosis of meningitis. The aim of the invention is to increase the sensitivity and simplify the method, as well as reduce the study time. For this, a 2% solution of agar on a 0.1 M biphthalate buffer or a 0.5% hypotonic solution of NACL with PH 6.2, a solution of polymyxin M sulfate in a concentration of 2-4 thousand U / ml, dry the mass of M. LYSODEIRTICUS test culture at a rate of 0.15% to the total volume of the medium, which is triturated in a mortar with 1-2 drops of buffer or hypotonic solution. An equal volume of polymyxin solution is added to the molten agar, the mixture is cooled to 44-55 ° C, after which the micrococcal culture is added and 4 ml are applied to slides. In a frozen medium, wells with a diameter of 2 mm are made at a distance of 1 cm from each other. The wells are filled with samples of cerebrospinal fluid, after which the slides are kept in a thermostat for 15-120 minutes. The presence of lysozyme is judged by the appearance of a lysis zone around the well during the specified observation period. If lysis is detected within 15-120 minutes, the bacterial etiology of meningitis is diagnosed, and in the absence of lysis,

Description

The invention relates to medicine, in particular medical microbiology, and can be used in the diagnosis of meningitis.

The aim of the invention is to increase the sensitivity and simplify

method, as well as reducing the time of the study.

The method is carried out as follows.

Prepare a 2% solution of agar from Bactoagar Difco or simple agar

agar of domestic production on O, 1 M biphthalate buffer or 0.5% hypotonic NaCl solution with a pH of 6.2. A solution of polymicin No. sulfate is prepared at a concentration of 2–4 thousand U / ml, for which a suspension of an antibiotic or a tablet containing 500 thousand U is taken and dissolved in 250–175 ml of buffer or saline. A portion of the dry weight of the M. lysodeikticus culture test is taken at a rate of 0.15% of the total volume of the medium and carefully ground in a mortar with 1-2 drops of buffer or hypotonic solution. An equal volume of the prepared polymyxin solution is added to the molten 2% agar, the mixture is cooled to a temperature of 44-55 ° C, after which it is combined with the prepared suspension of a micrococcal culture and applied to 4 ml on glass slides. In a frozen medium, wells with a diameter of 2 mm are made at a distance of 1 cm from one another.

Before the examination, the slides with medium are preheated in a thermostat for 15 to 20 minutes. The wells are filled with test samples of cerebrospinal fluid, after which the slides are kept in a thermostat for 15-120 minutes. The presence of lysozyme is judged by the appearance of a lysis zone around the well during a specified period of time. If lysis is detected within 15–120 min, the bacterial etiology of meningitis is diagnosed, and in the absence of lysis, viral one is detected for a specified time.

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Example 1. Patient N., 12 years old

admitted with a preliminary diagnosis: acute meningitis of unrecorded etiology. The patient produced spinal puncture. In the study of cerebrospinal fluid found: cerebrospinal fluid transparent, cytosis 198 106 cells / l, neutrophils 48%, lymphocytes 52%, protein 0.33 g / l, sugar 2.4 mmol / l chlorides 125 mmol / l. In order to establish the etiology of meningitis, the lysozyme activity of the cerebrospinal fluid of the patient has been studied. A gel-bacterial mixture was prepared for future use in 20 proposed studies. For a single calculation, 4 ml of the medium required for one slide was taken and the optimal number of possible

0

five

0

five

jO jj

40

45

50

55

research on it (2-3 samples of liquor). It took 28 NPs of the gel-bacterial mixture to complete the intended number of studies. 14 ml of a 2% solution of agar was prepared from simple domestic-produced agar-agar with a 0.5% solution of sodium chloride, pH 6.2. A solution of polymyxin M sulfate containing 4 thousand U / ml was prepared by dissolving 1 antibiotic tablet (500 thousand U) in 175 ml of 0.5% sodium chloride solution.

A portion of M. lysodeikticus dry culture (42 µg) was carefully ground in a mortar with 1-2 drops of hypotonic NaCl solution. To 14 ml of molten 2% agar was added 14 ml of the prepared solution of polymyxin Sulphate, the mixture was cooled to 50 ° C, and then combined with a suspension of micrococcus culture. The medium was poured into 4 ml on 7 glass slides. In a frozen medium, 3 wells were made at a distance of 1 cm from each other. Using a Pasteur pipette, the well was filled with the samples of the liquor examined, after which the slide was placed on the bottom of the Petri dish and viewed every 10–15 min for 2 h. The appearance of the lysis zone, visually perceived as an enlightenment of the medium around the well, began after 115 min , which allowed to make a conclusion about the bacterial nature of meningitis in this patient and prescribe etiotropic therapy. The results of bacteriological examination of the patient's spinal-mohegous fluid confirmed the meningococcal etiology of the disease on the third day,

PRI mme R 2. Patient K., aged 16, was hospitalized with a diagnosis of meningococcal disease meningitis. Medical data: cytosis 700x10 cells / l, neutrophils 55%, lymphocytes 45%, protein 0.48 mg / l, sugar 2.8 mmol / l, chlorides 115 mmol / l. The results of microscopy of the cerebrospinal smear are negative. Determination of the lysozyme activity of the cerebrospinal fluid was carried out according to the proposed method. The gel-bacterial mixture was prepared in the same way. in example I, except that the dose of polymyxin in the medium was 1 thousand U / ml. The course of the research is the same. The appearance of a lysis zone around a liquor well 5

It is noted after 15 minutes of incubation in a thermostat. The conclusion was made about the bacterial nature of the disease. Before the final diagnosis was made, in the nidus, a medical examination of the patient was conducted, patients with characteristic clinical signs of nasopharyngitis were diagnosed and isolated. On the 3rd day of bacteriological examination, meningococcus was isolated from the patient's nasopharynx, and in RIGA, on the 7th day, an increase in antibody titers to meningococcal polysaccharide was observed. Final diagnosis: meningococcal disease, meningitis. Quantitative determination of lysozyme in this patient, carried out in a known manner, showed its presence in a dose of 16 µg / ml.

The use of the proposed method allows to increase the sensitivity of diagnosis of meningitis by 30%, simplify the method due to the simplicity of preparing and accounting for the reaction and reduce the time of research to 2 hours.

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The table shows the number of detected lysozyme proposed and and by known methods.

Claims (1)

Invention Formula A method for the differential diagnosis of bacterial and viral meningitis, including the determination of lysozyme in a biological fluid by diffusion in an agar with M. 1-sodeicticus test culture, characterized in that, in order to increase the sensitivity, simplify the method and shorten the study, the - cerebral fluid, while 1-2 thousand U / ml of polymyxin is added to the agar; then the lysis zones of the medium are visually determined and, if lysis is detected, within 15-120 minutes a bacterial etiology is diagnosed meningitis is viral in the absence of lysis for a specified time. Editor V. Petrash Compiled by G. Chuich Tehred M. Didyk Order 39 Circulation 503 VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35; Raushsk nab. 4/5 Proofreader S.Yuekmar Subscription