RU2224257C1 - Method for predicting salmonellosis - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: the present method deals with detecting antibodies to Salmonella in blood serum due to microagglutination immunofluorescent reaction (MAIFR) with fluorescent complex and monogroup (to antigens of A, B, C, D, E) salmonellosis diagnostic kits supplied with Fab-fragments of immunoglobulins to heterologous microorganisms of intestinal group. If smears contain agglutinates containing 25 and more fluorescent Salmonella it is possible to diagnose salmonellosis. MAIFR is performed at the volume of not less than 2 mcl beginning with whole dilution of blood serum. EFFECT: higher sensitivity and specificity of diagnostics. 2 cl, 6 ex, 1 tbl

Description

 The invention relates to medicine, namely to microbiology and immunology, and can be used in medical practice for the detection of antibodies to Salmonella groups A, B, C, D, E in adults and children.

 In applied immunology, practical and scientific, and bacteriological work, testing of antibodies to Salmonella groups A, B, C, D, E is carried out in RA, RNGA.

 Birger M.O. Handbook of microbiological and virological research methods - M .: Medicine, 1982, -462 p.

 Labinskaya A.S. Microbiology with the technique of microbiological research methods. - M .: Medicine, 1988, - 440 p.

 Pokrovsky V.I., Kilesso V.A., Yushchuk N.D. Salmonellosis. - Tashkent: Medicine, 1989, - 344 p.

 A known prototype method for detecting antibodies to salmonella obtained by the method protected by patent 2175558 of 11/27/2000, which consists in the use of immunofluorescence microagg-lutination (IFRMA) fluorescence complex and single-group diagnostics containing normal rabbit serum in the vehicle.

 According to the prototype method, a standardized and controlled suspension of fluorescent complex and monogroup salmonella diagnostics contains normal rabbit serum to a final concentration of 0.1%.

 The disadvantage of using the prototype method is nonspecific reactions in IFRMA with sera of patients containing antibodies to heterogeneous pathogens of the intestinal group, due to the presence of cross-reactive determinants in salmonella and intestinal group of microorganisms. This makes impossible the reliable determination of antibodies in the initial dilutions of blood serum at their low titers (see table).

 The invention is aimed at solving problems: increasing the sensitivity and specificity of the method; detection of specific antibodies in low dilutions of serum, starting from whole; reducing the cost of diagnostic kits.

 These tasks are achieved through the use of IFRMA, characterized in that the reaction is carried out with fluorescent complex and monogroup diagnostics, in which Fab fragments of immunoglobulins to heterologous microorganisms of the intestinal group are introduced instead of normal rabbit serum. The latter interact with the cross-reactive determinants of the diagnosticum and block them, ensuring that only specific determinants of the diagnosticum interact with the corresponding antibodies to salmonella in the reaction with the formation of agglutinates. The resulting complexes of antibodies and fluorescent complex and monogroup salmonella diagnostics are examined in a luminescent microscope and, in the presence of agglutinates containing 25 or more fluorescent salmonella, salmonella is diagnosed. The IFRMA reaction is delivered in a volume of at least 2 μl, starting with a complete dilution of blood serum.

 The method is as follows. Lyophilized fluorescent complex and monogroup salmonella diagnostics containing 0.1% Fab fragments of immunoglobulins to heterologous microorganisms of the intestinal group obtained from agglutinating Escherichia OK sera are used for formulation.

IFRMA with dry diagnostics is carried out as follows: on slides (divided into 8 squares) placed in an open wet chamber, apply 2 μl of successive dilutions of test sera from whole to 1: 640. Then, 2 μl is added to each drop of serum dilutions group A fluorescent salmonella diagnosticum, the droplets are mixed and the chamber is closed. Then the chamber with the slides is placed in a thermostat (t = 37 ^° C), where it is kept for 60 minutes. After that, the chamber is opened, the droplets on the glasses are dried and the preparations are examined in the field of view of a luminescent microscope. A similar formulation is carried out with fluorescent salmonella diagnostics of other groups B, C, D, E and complex.

 In the presence of specific antibodies (to a salmonella homologous type) in the serum, the formation of agglutinates containing fluorescent salmonella is observed.

 Evaluation of the results is carried out by the number of salmonella in the agglutinate and the presence of separately located salmolnella.

The results are taken into account on an alternative assessment scale, provided that 8-10 fields of view are viewed:
+ visual fields are filled with large and medium-sized agglutinates (25 or more salmonella in one agglutinate) and single separately located salmonella;
- the visual fields are filled with small agglutinates (less than 25 salmonella in one agglutinate) and a large number of separately located salmonella or salmonella in all fields of vision are located separately.

 Serum titer is considered its maximum dilution, which causes agglutination of the diagnosticum with a content of 25 or more fluorescent salmonella in one agglutinate. If agglutinates of the indicated size are present in the test serum, salmonellosis is diagnosed.

The reaction should be accompanied by the following controls:
1. Obviously positive control: standard diagnostic serum + fluorescent salmonella diagnostics, serum titer should be confirmed.

 2. A known negative control: serum that does not contain specific antibodies + fluorescent salmonella diagnostics. Agglutinates are absent.

 3. Control of diagnostics: saline + fluorescent diagnostics. Agglutinates are absent.

 The duration of the reaction is 1 h 10 min.

Examples of specific performance
Example 1

 Patient D., 11 years old, was admitted to the clinic on the 1st day of illness. Ill acutely. In the morning there was nausea, malaise, then vomiting, which appeared twice a day, there was no loose stool.

Upon admission, a moderate condition, temperature 38.5 ^o C. In consciousness. The skin is clean, skin elasticity and turgor are preserved. Pulse 108 beats in 1 min. The tongue is dry, covered with a gray-white coating. The abdomen on palpation is soft, painful in the epigastric and ileal regions, rumbling along the large intestine. The liver is at the edge of the costal arch, the spleen is not palpable. Diuresis saved. There was no stool at the reception, from the first day in the department it was watery, yellow, without pathological impurities, then gruff, decorated from 6 days. Subfibrilally temperature for 7 days.

In the general analysis of blood, leukocytosis is 10.3 • 10 ⁹ g / l, neutrophilia with a left shift to p / I - 29%, significantly accelerated ESR - 39 mm / h.

 Urinalysis unchanged.

 In the coprogram, mucus +++, undigested carbohydrates ++.

 Tank. stool analysis 3 times negated.

 Serological studies.

 In IFRMA with a fluorescent diagnosticum obtained by the prototype method, agglutinates were found containing 30-45 fluorescent Salmonella in a dilution of 1: 320 (titer 1: 320). Nonspecific reactions were revealed at a 1:80 dilution with pathogens of the intestinal group (Escherichia).

 In IFRMA with a fluorescent diagnosticum with Fab fragments obtained by the claimed method, agglutinates containing 30-50 fluorescent salmonella in a dilution of 1: 320 (titer 1: 320) were recorded, starting with a complete dilution of serum. Nonspecific reactions with Escherichia were not observed.

 The results are confirmed by control studies of serum in RNGA with complex erythrocyte salmonella diagnosticum A, B, C, D, E (titer 1: 320).

 Diagnosed with salmonellosis, gastrointestinal form (gastroenterocolitis) of moderate severity, acute course. Discharged in satisfactory condition.

Example 2
Patient M., 1 year. 8 months, was admitted to the clinic on the 6th day of illness with complaints of fever up to 39 ^o C, vomiting up to 3 times a day, loose stools with herbs and mucus up to 5 times a day. She became acutely ill with fever up to 38 ^o C, the appearance of loose stools. They were treated on an outpatient basis with furazolidone, smecta, paracetamol - without effect. Sent to a hospital.

 The condition at receipt of moderate severity, sluggish, decreased appetite, skin is pale, skin elasticity and turgor are preserved. The tongue is wet, clean. The abdomen is soft, painless. The liver is at the edge of the costal arch, the spleen is not palpable. Diuresis saved. The stool is liquid, yellow, with a large amount of muddy mucus for 4 days in the hospital, then gruel, and after 7 days - decorated.

In OAK, white blood cells - 6.8 • 10 ⁹ g / l, s / I - 5%, s / I - 30%, l - 54%, m - 10%, e - 1%, ESR - 22 mm / h, moderately accelerated .

 In OAM, leukocytes 4-6 P / sp., Protein - neg., Bacteria +, phosphates +++.

 Tank. fecal analysis is 4 times negative.

 Serological tests. In IFRMA on the 14th day of illness with a fluorescent diagnosticum obtained by the prototype method, agglutinates were found containing 35-50 fluorescent salmonella in a dilution of 1: 1280 (titer 1: 1280). Nonspecific reactions with Escherichia titer 1:40 were noted.

 In IFRMA with fluorescent diagnosticum and Fab fragments obtained by the claimed method, agglutinates were found containing 40-50 fluorescent salmonella in a dilution of 1: 1280 (titer 1: 1280). There are no reactions with Escherichia, starting with a complete dilution of serum.

 The results are confirmed by control studies of serum in RNGA with complex erythrocyte salmonella diagnosticum A, B, C, D, E (titer 1: 1280).

 Diagnosed with salmonellosis, gastroenterocolitic form, moderate severity (clinically and serologically), acute course. The patient was discharged from the hospital in satisfactory condition.

Example 3
Patient K., 7 years old, was admitted to the clinic on the 2nd day of illness with complaints of fever up to 39 ^o C, abdominal pain, vomiting, loose stool up to 3-4 times a day. Condition upon receipt of moderate severity, temperature 37.4 ^o C, lethargic, reduced appetite, skin clean, pale, tongue moist, lined with gray-white bloom. The abdomen on palpation is soft, painful along the large intestine, the liver is 2 cm below the edge of the costal arch, the spleen is not palpated. The chair at the reception and the next 3 days in the department is liquid, with greens and mucus.

In the general analysis of blood, leukocytes 8.1 • 10 ⁹ g / l, neutrophilia with a shift to the left to p / I - 42%, ESR - 15 mm / h.

 In the general analysis of urine, no changes were detected.

 Tank. stool analysis is 4 times negative.

 Serological analyzes: in IFRMA on the 7th day of illness with a fluorescent diagnosticum obtained by the prototype method, agglutinates were found containing 25-40 fluorescent salmonella in a dilution of 1: 640 (titer 1: 640). Nonspecific reactions were observed at a dilution of up to 1:80 with intestinal pathogens (Escherichia).

 In IFRMA with fluorescent diagnosticum and Fab fragments obtained by the claimed method, agglutinates containing 32-65 fluorescent salmonella in a 1: 640 dilution (titer 1: 640) were observed. Nonspecific reactions with Escherichia were not noted, starting with a complete dilution of serum.

 The results are confirmed by control studies of serum in RNGA with complex red blood cell salmonella diagnosticum A, B, C, D, E (titer 1: 640).

 Diagnosed with salmonellosis, gastroenterocolitic form, moderate (clinically and serologically), acute course. Discharged from the department on the 11th day in satisfactory condition.

Example 4
Patient A., 4 years old, was admitted to the hospital with complaints of fever up to 39 ^o C, repeated vomiting, loose, watery stools on the 2nd day of illness. Ill acutely. Condition upon receipt of moderate severity, temperature 37.9 ^o C. The skin is pale with a marble pattern, skin elasticity and turgor are preserved. The tongue is dry, coated with a gray-white coating. Tachycardia up to 120 per minute. The abdomen on palpation is soft, painful in the left ileal region, the sigmoid colon is spasmodic.

In the general analysis of blood, leukocytosis is 12.6 • 10 ⁹ g / l, neutrophilia with a left shift to p / I is 40%, accelerated ESR up to 25 mm / h.

 In the coprogram, mucus, undigested carbohydrates, bacteria.

 Tank. stool analysis 3x neg.

 Serological tests.

 In IFRMA with a fluorescent diagnosticum obtained by the prototype method, agglutinates were found containing 25-40 fluorescent salmonella in a dilution of 1: 160 (titer 1: 160). Non-specific reactions were diluted to 1:20 with intestinal pathogens (Escherichia).

 In IFRMA with fluorescent diagnosticum and Fab fragments obtained by the claimed method, agglutinates containing 30-60 fluorescent salmonella in a 1: 160 dilution (titer 1: 160) were observed. Nonspecific reactions with Escherichia were not noted, starting with a complete dilution of serum.

 The results are confirmed by control studies of serum in RNGA with complex erythrocyte salmonella diagnosticum A, B, C, D, E (titer 1: 160).

 Diagnosed with salmonellosis, gastroenterocolitic form, moderate severity (clinically and serologically), acute course. Discharged from the department on the 11th day in satisfactory condition.

Example 5
Patient B., 1 g. 3 months, was admitted to the clinic on the 2nd day from the onset of the disease with complaints of abdominal pain, frequent loose stools, fever up to 38 ^o C. She became ill sharply. An ambulance doctor was hospitalized in the children's surgical department to exclude acute appendicitis due to severe abdominal pain, where she was observed during the day.

In the general blood test, leukocytosis was observed up to 16.3 • 10 ⁹ g / l, p / I - 38%, ESR - 40 mm / h.

During the observation, the pain decreased, the diagnosis of acute appendicitis was withdrawn and the girl was transferred to our clinic with acute intestinal infection. Condition upon receipt of moderate severity. Skin and visible mucous membranes of a physiological color, moist. Turgor tissue preserved. Tachycardia up to 124 per minute against a background of a temperature of 38 ^o C. In the oropharynx moderate hyperemia of the arches and tonsils. Scleritis. The tongue is covered in gray and white. The abdomen is soft, painless, the liver 1 cm, the spleen is not palpable. Diuresis saved. The stool on the first day in the hospital up to 5 times a day, liquid, green, with mucus and streaks of blood, from 3 days - mushy, from 5 days - decorated.

In the general analysis of blood, leukocytosis was replaced by leukopenia up to 5.7 • 10 ⁹ g / l, s / I - 10%, lymphocytosis, ESR - 2 mm / h.

 In a coprogram, an increase in white blood cells is up to 15-20 in n / a., Undigested carbohydrates +, bacteria +, red blood cells 10-15 in n / a.

 Tank. stool analysis 3 times negated.

 Serological studies.

 In IFRMA with a fluorescent diagnosticum obtained by the prototype method, agglutinates were found containing 35-40 fluorescent Salmonella in a dilution of 1: 320 (titer 1: 320). Non-specific reactions were observed in dilutions up to 1:20 with intestinal pathogens (Escherichia).

 In IFRMA with fluorescent diagnosticum and Fab fragments obtained by the claimed method, agglutinates containing 28-55 fluorescent salmonella in a dilution of 1: 320 (titer 1: 320) were observed. Nonspecific reactions with Escherichia were not noted, starting with a complete dilution of serum.

 The results are confirmed by control studies of serum in RNGA with complex erythrocyte salmonella diagnosticum A, B, C, D, E (titer 1: 320).

 The results are confirmed by control studies of serum in RNGA with complex erythrocyte salmonella diagnosticum A, B, C, D, E, in which the titer is 1: 320, the test with unitiol is negative.

Diagnosed with salmonellosis (clinically and serologically), enterocolitic form, moderate, acute course. Discharged in satisfactory condition
Example 6
Patient A., 2 years, 2 months, was admitted to the hospital with complaints of fever up to 38 ^o C. Before that, for 2 weeks she was treated on an outpatient basis for acute respiratory viral infections by symptomatic means. Condition upon receipt of moderate severity, temperature 38.3 ^o C. The skin is pale, clean. Mucous wet. The pharynx is hyperemic. From the nose, mucous discharge. The tongue is clean, moist. The abdomen is soft, painless. Liver +2 cm, spleen not palpable. Diuresis saved. There was no stool. In the department in the morning and on the 1st day of admission, loose stools, dark brown with streaks of mucus and streaks of blood. From the anamnesis it became known that on the eve of the disease fed cottage cheese with sour cream, bought in a store. Subsequently, the stool in the department is liquid, watery with flakes of cloudy mucus up to 7 times a day for 2 days, then gruel and decorated.

When examining in OAK, leukocytosis is up to 12.1 • 10 ⁹ g / l, neutrophilia with a left shift to p / I - 18%, ESR - 7 mm / h. Feces on worm eggs and protozoa are negative. Tank. stool analysis is 3 times negative. Feces for viruses - no rotaviruses have been detected.

 Serological studies.

 In IFRMA with a fluorescent diagnosticum obtained by the prototype method, agglutinates containing 8-10 fluorescent Salmonella were detected in a 1:20 dilution, i.e. the results were negative. Non-specific reactions with Escherichia at a 1:20 dilution were noted.

 In IFRMA with a fluorescent diagnosticum and Fab fragments obtained by the claimed method, no agglutinates of fluorescent Salmonella were observed, starting with a complete dilution of serum.

 In RNGA with a complex erythrocyte salmonella diagnosticum A, B, C, D, E, the antibody titer was 1:10, and when inhibiting RTNGA with a specific antigen, no inhibition of reaction was observed, which indicated non-specific results obtained in RNGA.

 Diagnosed with infectious enterocolitis of unknown etiology, moderate severity, acute course. Discharged in satisfactory condition.

 The positive effect of this technique is to increase the sensitivity and specificity compared to the prototype method, the ability to register antibodies in whole serum, which is important for the low antibody titers observed in children, as well as a 2-fold reduction in the economic costs of diagnostics

Claims (2)

1. A method for the diagnosis of salmonellosis using immunofluorescence microagglutination reaction (IFRMA) by the interaction of specific antibodies of the blood serum with fluorescent complex and monogroup salmonella diagnostics, characterized in that Fab - fragments of immunoglobulins to heterologous microorganisms are introduced into the diagnostics, in the presence of heterologous microorganisms and microorganisms agglutinates containing 25 or more fluorescent salmonella are diagnosed with salmonellosis. 2. The method according to claim 1, characterized in that the IFRMA reaction is set in a volume of at least 2 μl, starting with a complete dilution of blood serum.

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