RU2425384C2 - Diagnostic technique for clinical course of salmonellosis-protozoal acute enteric infections in children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: faeces sample is examined in patients with acute enteric infections by bacteriological and microscopical methods. One faeces sample of the patient is concentrated by acetate sedimentation, and the produced sediment is used to prepare simultaneously 3 smears: the first one is stained with Ziehl-Nielsen carbolic fuchsin for acid-fast intestinal protozoa indication, the second one is stained with 1% Lugol's solution for lamblia detection, the third one is stained with polyvalent adsorbed serums in an indirect immunofluorescence reaction for salmonella indication, and is observing specific luminescence, the salmonellosis-protozoal acute enteric infection is diagnosed. 1-4 cells in a field of view enable to diagnose a mild form, and 5-12 cells - a severe infection.

EFFECT: use of the declared method allows producing results already in 1-1,5 h after sampling the material and providing higher diagnostic accuracy ensured by quantitative analysis of adhesive activity of simultaneously examined faeces samples, both for salmonellas, and for intestinal protozoa.

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Description

The invention relates to medicine, in particular to microbiology and infectious diseases, and can be used to diagnose various forms of salmonella protozoan acute intestinal infections in children.

The particular urgency of the problem of salmonellosis is determined by the continued increase in their specific gravity in the infrastructure of acute intestinal infections (ACI). The predominant salmonellosis of children, especially young children, the widespread prevalence, severity and nature of the infection process in salmonellosis, often taking a protracted and recurrent course, determine the priority of studying the problem.

In a significant number of cases, salmonellosis proceeds as a mixed infection. According to different authors, mixed forms account for up to 70%. In the structure of associates, with mixed forms of salmonella, along with other types of enterobacteria and viruses, intestinal protozoa occupy a leading place. Often, such a combination of pathogens causes a distortion of typical clinical symptoms, and also makes standard antibiotic therapy regimens ineffective or ineffective, which generally significantly worsens the outcome of the disease. In this regard, the improvement of the etiological decoding of combined salmonella protozoal forms of the disease, such as salmonella giardiasis, salmonella cryptosporidiosis, salmonella amoebiasis, etc., aimed at a more complete identification of protozoal invasion in children with protracted forms of salmon adequate therapy is an urgent and urgent problem of infectology.

Known methods for the diagnosis of mixed forms of OCI based on the assessment of the adhesive activity of pathogens in mixed infections of various etiologies. So, there is a known method for the diagnosis of mixed forms of OCI caused by Klebsiella spp., Citrobacter spp., Rettgerella spp. in association with Proteus vulgaris, based on the identification of adhesins by searching for target cells and elucidating the role of organelles that determine the degree of adhesive activity of bacteria of the genus Proteus in the hemagglutination reaction (ZG Gabidullin, IB Ishkildin. Bashkir Medical Institute of the Ministry of Health of the RSFSR, Ufa Scientific Research Institute of Vaccines and Serums named after II Mechnikov of the Ministry of Health of the USSR, Ufa // Collected Scientific Papers “Mixed Infections”, Scientific Research Institute of Epidemiology and Microbiology named after NF Gamalei, M., 1986).

However, the proposed method for the diagnosis of mixed bacterial-proteic OCI, based on the indication of protea adhesins, only indirectly indicates the etiological role of protea in the development of mixed infection in the intestine, because the method does not allow to identify the true mechanism of interaction of strains of OKA pathogens, such as Klebsiella spp., Citrobacter spp., Rettgerella spp. with Proteus vulgaris.

There is also a method for the diagnosis of OCI of conditionally pathogenic and mixed etiology (Diagnosis, prognosis of the course and treatment of acute intestinal infections of conditionally pathogenic and mixed etiology // Met. Recommend., Leningrad Research Institute of Children's Infections, Leningrad, 1990), based on the complex consistent bacteriological and serological studies providing registration of the increase in the titer of UPM antibodies (the level of specific antibodies in RA with an auto-strain or RPHA with an erythrocytic diagnosticum), together with a wedge cal symptoms, testifying to the AEI in children, caused by UPM, and aimed to confirm the significance of the selected micro-organisms and their associations.

However, the above method for the diagnosis of acute intestinal infections of mixed etiology concerns only bacterial-bacterial combined infections and does not affect relationships in bacterial-protozoal associations, and is also very time-consuming and time-consuming for diagnosis and does not provide accuracy.

Currently, no sources of a method for diagnosing protozoal-bacterial OCI are available to us.

Closest to the proposed method is a diagnostic method for mixed forms described in MU No. 04-723 / 3 dated 12/17/1984, “Methodological guidelines for microbiological diagnosis of diseases caused by enterobacteria,” and MUK 4.2.735-99 “Parasitological methods for laboratory diagnostics” helminthiases and protozoa, "2000. According to this method, the diagnosis of salmonella infection is carried out mainly using bacteriological studies using standard methods for the isolation and identification of salmonella. The study involves the isolation of salmonella in a bacteriological laboratory. In this case, the study of the patient's feces for intestinal protozoa is carried out in a clinical laboratory according to the generally accepted method. Thus, the main disadvantage of the above method is the study of fecal samples from a patient with suspected salmonella-protozoal acute intestinal infections in various laboratories: bacteriological for the isolation of salmonella and clinical for protozooscopy. Moreover, studies of salmonellosis and protozoa samples are not always carried out from one portion of the patient's feces. Such an algorithm for conducting research does not provide evidence of the mixed nature of the salmonella protozoal OCI, does not have sufficient effectiveness and accuracy.

In order to eliminate the above drawbacks, the authors proposed a fundamentally new method for diagnosing the course of salmonella protozoan acute intestinal infections.

The technical result consists in increasing the accuracy of diagnostics by quantifying the adhesive activity of simultaneously tested stool samples for both salmonella and intestinal protozoa.

This result is achieved by the fact that in the known method, which includes the study of stool samples in patients with acute intestinal infections by the bacteriological method for salmonella and the microscopic method for protozoal cysts, according to the invention, the stool sample is concentrated, 3 smears are prepared from the obtained suspension, simultaneously for 1-1 , 5 hours stained with carbol fuchsin according to Ziehl-Nielsen to indicate acid-resistant intestinal protozoa, 1% Lugol's solution for the detection of lamblia, polyvalent adsorbed serums in the reaction did not direct immunofluorescence for the indication of salmonella and in the presence of a specific luminescence of salmonella on the surface of protozoal cysts, a mixed etiology of salmonella-protozoan OCI is established, and a mild form, from 5–4 cells, is diagnosed by the number of salmonella-adherent cells on cysts in the field of view of 1–4 - protracted form of infection.

The proportion of detection and evidence of the etiological significance of protozoan cysts in association with salmonella using the method proposed by the authors was 89%, while the identification of associates using the prototype method of scattered detection at the bases of two different laboratories, in comparison with the author's method, was only 15% ( see drawing).

For the first time, we offer the simultaneous preparation of smears for staining both on acid-resistant intestinal protozoa and lamblia, as well as on salmonella.

We propose a preliminary concentration and enrichment of a sample of feces by ether-acetic sedimentation of protozoal cysts. At the same time, we found the achievement of the highest concentration of cysts necessary for further study of the Salmonella sample.

For the first time, we established the ability of Salmonella to exhibit adhesive activity to the surface of protozoan cysts, which is the main mechanism for the development of the infectious process, similar to the mechanism of adhesion to cells of non-phagocytic epithelium, as well as free cells of multicellular organisms. The authors found that the detection of salmonella adhering to the surface of protozoan cysts confirms the mixed nature of the infection during the salmonella protozoal process in the intestine, since the staining and washing steps during the formulation of the indirect immunofluorescence reaction completely exclude the possibility of non-specific nature of salmonella adhesion to cysts.

The difference between the method is the fact that the diagnosis of salmonella protozoal infection takes 1-1.5 hours, in contrast to the closest prototype method, in which the indication of salmonella and intestinal protozoa requires 24-48 hours. We use salmonella adsorbed polyvalent ABCDE serum for staining, which confirms the specificity of the glow in the reaction of indirect immunofluorescence to salmonella.

For the first time, the authors propose a quantitative accounting of intestinal protozoa salmonella adhered to cysts and diagnose a mild degree of salmonella adherent to cysts in a field of view of 1–4; mild infection from 5–12 salmonella cells, thereby predicting the degree of mixed infection the severity of the course of protracted forms of salmonella protozoal acute intestinal infections.

The proposed method is as follows. Samples of feces from patients with acute intestinal infections of salmonella etiology, delivered to the laboratory, are subjected to ether-vinegar sedimentation to achieve the highest concentration of cysts and trophozoites of intestinal protozoa that are supposed to be in them. Three smears are simultaneously prepared from the resulting precipitate. A drop of a 1% aqueous Lugol solution is immediately added to one drug for the differential diagnosis of amoeba and flagellum protozoa, including lamblia and dientameba. Microscope, noting the adhesion on the surface of cysts of small sticks.

The other two drugs are dried at room temperature, then fixed in cold acetone and stained with carbol fuchsin according to Ziehl-Nielsen for the detection of cryptosporidium oocysts and isospores. In case of detection of red colored oocysts, adhesion on their surface of small, green-colored sticks is noted. The remaining preparation is tested in an indirect immunofluorescence reaction using diagnostic adsorbed polyvalent salmonella O-serum, main groups A, B, C, D, E. A drop of rabbit adsorbed polyvalent salmonella O-serum A, B, C, D is applied to a fixed smear. E. Then the drug is tinted with antispecies luminescent serum with simultaneous contrasting. The specificity of adhesion is confirmed by the stages of staining and washing during the formulation of the indirect immunofluorescence reaction and completely eliminates the possibility of a nonspecific nature of the adhesion of salmonella to cysts.

A positive specific result is a bright specific glow (fluorescence) with a greenish light around the periphery of the cysts (in the area of salmonella adhesion on the surface), clearly contrasting with the dark body of intestinal protozoa cysts. The morphology of the species, as a rule, is preserved. Evaluation of the drug should be carried out comprehensively taking into account indicators such as brightness and color of specific fluorescence, localization and structure of the glow, as well as the number of protozoan salmonella adhered to cysts in the field of view.

To establish the form of the disease, the average value of salmonella cells adhered to the surface of the cysts is calculated in the field of view. For this, under microscopy under a luminescent microscope, at least 3 fields of view are examined, in each of which cysts and salmonella adhered to them are counted. Then calculate the average value of the adherent cells. With a value of 1-4, a mild form is diagnosed, from 5-12 Salmonella cells, a protracted form.

To conduct a full range of research on the author’s method and diagnose salmonella protozoal infection, 1-1.5 hours are spent, in contrast to the closest prototype method, in which the indication of salmonella and intestinal protozoa requires 24-48 hours.

Thus, the advantage of the proposed method for diagnosing the course of salmonella protozoan acute intestinal infections in children is its high sensitivity and specificity, simplicity, accessibility, diagnostic accuracy, the possibility of obtaining results after 1-1.5 hours after taking the material, and the use of the method as in the early diagnosis of clinical forms of salmonella protozoal infection, and in examinations of patients with salmonella carriage.

The inventive method can be confirmed by the following examples.

Example 1

Patient S., 5 years 2 months. Diagnosis: salmonellosis typhimurium var. copenhagen, severe, acute course.

Options Indicators before the study on a method for diagnosing the nature of the course of salmonella protozoal infection in children Indicators as a result of changes in treatment tactics after applying the method for diagnosing the nature of the course of salmonella protozoal infection in children 1. Dates of admission to the hospital 7 days of illness - 2. Bed days 20 days - 3. Bacterial excretion 28 days 4. The diagnosis On admission: salmonellosis typhimurium var. copenhagen, severe, acute Ultimate: mixed intestinal infection. Salmonellosis. Giardiasis Gastrointestinal protracted form. Acute course. Severe degree 4 Presence of DZHVP Symptoms of DZhVP and changes in the gallbladder and bile ducts on ultrasound Symptoms of JVP with less intensity and frequency, the disappearance of changes in ultrasound 5. Clinical blood test Eosinophils 12-14 Eosinophils 1-2 ESR up to 34 mm / hour ESR 4 mm / hour Hb increased to 135 g / l HB 110 g / l Leukocytosis up to 9 White blood cells 2 Monocytes up to 7-9 Monocytes 1-2 6. Coprogram FATS: FATS: neutr. fats up to 3 neutr. fats 1 washed. sour up to 2 washed. sour up to 2 soap up to 2 soap 1 A lot of mucus White blood cells 2-3 White blood cells cover the entire field of view (50-60), neutrophilic Prismatic epithelial cells in p / z are absent Prismatic epithelial cells in p / z 7. Clinical manifestations: pain along the intestines, fever, symptoms of intoxication, impaired stool, vomiting + - 8. Indicators of a method for diagnosing the nature of the course of salmonella protozoal infection in children Massive seeding with Giardia cysts The absence of cysts in smears with salmonella adhered to them The average value of cysts adhered to the surface in p.z. 10

As can be seen from the above data, after the author’s methodology was carried out, changes were made to the diagnosis, a mixed salmonella-giardiasis infection was introduced, the etiotropic therapy strategy was changed, which determined the normalization of laboratory parameters and the patient’s clinical recovery.

Example 2

Patient K., 2 months Diagnosis: salmonellosis typhimurium, moderate severity, gastrointestinal form.

Options Indicators before the study on a method for diagnosing the nature of the course of salmonella protozoal infection in children Indicators as a result of changes in treatment tactics after applying the method for diagnosing the nature of the course of salmonella protozoal infection in children 1. Dates of admission to the hospital 9 days of illness - 2. Bed days 19 days - 3. Bacterial excretion 20 days - 4. The diagnosis On admission: salmonellosis typhimurium, moderate severity, gastrointestinal form Ultimate: mixed intestinal infection. Salmonellosis. Giardiasis Gastrointestinal form. Acute course. Severe degree 4 Presence of DZHVP Symptoms of DZhVP and changes in the gallbladder and bile ducts on ultrasound Symptoms of JVP with less intensity and frequency, the disappearance of changes in ultrasound 5. Clinical blood test Eosinophils 3 Eosinophils 1-2 ESR up to 32 mm / hour ESR 4 mm / hour Hb increased to 94 g / l HB 105 g / l Leukocytosis up to 10-13 White blood cells 2 Monocytes up to 5 Monocytes 1-2 6. Coprogram FATS: FATS: neutr. fats up to 3 neutr. fats 1 washed. sour up to 2 washed. sour up to 2 soap up to 2 soap 1 A lot of mucus White blood cells 2-3 White blood cells cover the entire field of view (50-60), neutrophilic Prismatic epithelial cells in p / z are absent Prismatic epithelial cells in p / z 7. Clinical manifestations: pain along the intestines, fever, symptoms of intoxication, impaired stool, vomiting + - 8. Indicators of a method for diagnosing the nature of the course of salmonella protozoan infection in children Massive seeding with Giardia cysts The absence of cysts in smears with salmonella adhered to them - The average value of cysts adhering to the surface in item 14

As can be seen from the above data, clinical and diagnostic parameters in the group of children with salmonella protozoan mixed infection are expressed in a distortion of typical clinical manifestations of both salmonella and giardiasis infections, manifesting themselves as a qualitatively new infectious disease, and are characterized by a longer and wavy course of acute intestinal infections statement of the author’s methodology, changes were made to the diagnosis, a mixed salmonella-giardiasis infection was introduced, the etiotropic strategy was changed therapy, which determined the normalization of laboratory parameters and the clinical recovery of the patient.

The proposed method was tested on material (stool samples) from 110 patients with acute intestinal infections (salmonellosis). The results of a study of the nature of the course of salmonella protozoan mixed infection with a quantitative assessment of salmonella cells adhering to the surface of protozoal cysts revealed that with a quantity of salmonella adhered to cysts of 1-4, a mild degree occurs, and with an amount of 5 to 12 - a severe degree of salmonella protozoan mixed infection, which correlated with the severity of the condition and the length of hospital stay of children. The clinical picture in patients with mixed protozoal infection, in contrast to patients with monosalmonellosis, was characterized by:

1) at a later date in hospital, with an increase in bed days;

2) an increase in the period of salmonella bacterial excretion, often turning into bacterial carriage;

3) admission to the hospital against the background of registration of the course of concomitant diseases;

4) severe symptoms of biliary dyskinesia and specific changes in the gallbladder and liver on ultrasound;

5) a history of allergic reactions;

6) a longer febrile period with subfibrillar numbers;

7) more severe exsicosis syndrome with toxicosis (I, II art);

8) an increase in the multiplicity of vomiting, with a longer period of their registration;

9) a large number of children with violations of the microstructure of the lumen of the colon, as well as earlier periods of development of intestinal dysbiosis;

10) more pronounced qualitative and quantitative changes in intestinal dysbiosis;

11) specific changes in the analysis results.

Thus, clinical and diagnostic features in the group of children with salmonella protozoan mixed infections are expressed in a distortion of typical clinical manifestations of both salmonella and protozoal infections, manifesting themselves as a qualitatively new infectious disease, and are characterized by a longer and wavy course of acute intestinal infections.

The method developed by the authors can be recommended for widespread use in practical health care for the diagnosis of the course (mild, severe) of salmonella protozoan acute intestinal infections (ACI) in children. The method has high sensitivity and specificity, simplicity and accessibility. The possibility of obtaining results within 1-1.5 hours after taking the material, as well as the use of the method both in the early diagnosis of the clinical forms of salmonella protozoal infection and in the examination of patients with salmonella carriage, makes it of great practical importance.

Claims (1)

A method for diagnosing the course of salmonella protozoal infection in children by examining fecal samples in patients with acute intestinal infections using a bacteriological and microscopic method, characterized in that one patient's feces sample is preconcentrated by ether-acetic sedimentation, three smears are prepared simultaneously from the precipitate: the first one - stained with carbol fuchsin according to Ziehl-Nielsen to indicate acid-resistant intestinal protozoa, the second - 1% Lugol's solution to detect giardia, the third - watering tnym adsorbed sera in indirect immunofluorescence reaction, for indicating detection of Salmonella and Salmonella-specific luminescence-diagnosed acute intestinal protozoal infection, and if the number in the field of view is 1-4 cells - diagnose mild, from 5-12 cells - severe infection.

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