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

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.

2 ex, 1 dwg

 

The invention relates to medicine, in particular to Microbiology and infectious diseases, and can be used for the diagnosis of various forms of Salmonella-acute intestinal protozoal infections in children.

Of particular relevance to the problem of Salmonella is determined by the continued growth of their share in the infrastructure of acute intestinal infections (AII). Primary defeat of salmonellosis in children, especially in the early age, the widespread prevalence, severity and nature of infection with Salmonella, often receiving prolonged and relapsing course, determine the priority of investigation.

In a significant number of cases of salmonellosis occur in the type of mixed infections. The share of mixed forms, according to different authors, accounting for up to 70%. In the structure associated with, the mixed form of Salmonella, along with other types of entero-bacteria, and viruses, the leading place is occupied by intestinal protozoa. Often this combination of pathogens causes distortion typical clinical symptoms, and also makes inefficient or ineffective standard antibacterial therapy that, in General, significantly affects the outcomes. In this regard, the improvement of the etiological decryption combined Salmonella-protozoal forms of ill the tion, such as salmonellosis giardiasis, salmonellosis-cryptosporidia, Salmonella-nebesna etc. aimed at a more thorough identification of protozoan infestations in children with persistent forms of Salmonella and further carrying out of adequate therapy is topical and urgent problem of Infectology.

Known methods of diagnosis of mixed forms of OKA based on the evaluation of the adhesive activity of pathogens in mixed infections of different etiology. So, there is a method of diagnosis of mixed forms of OKA, due to Klebsiella spp., Citrobacter spp., Rettgerella spp. in Association with Proteus vulgaris, based on detection of adhesins by finding the target cells and elucidate the role of organelles, determining the extent of the adhesive activity of bacteria of the genus Proteus in the reaction of haemagglutination (Schgublin, Ibisbills. Bashkir medical Institute of the Ministry of health of the RSFSR, Ufa scientific research Institute of vaccines and sera them. Mechnikov, USSR Ministry of health, Ufa // Sat. scientific papers "Mixed infection", research Institute of epidemiology and Microbiology. Nframe, M., 1986).

However, the proposed method for the diagnosis of mixed bacterial-proteaceae OKA, based on the indication of adhesins Proteus, only indirectly testifies to the etiologic role of Proteus in the development of mixed infection in the intestine, because the method does not reveal the true mechanistically strains, OKA, such as Klebsiella spp., Citrobacter spp., Rettgerella spp. with Proteus vulgaris.

There is also known a method for the diagnosis of OKA opportunistic and mixed etiology (Diagnosis, prognosis of and treatment of acute intestinal infections opportunistic and mixed etiology // Met. REC., The Leningrad scientific research Institute of children's infections, Leningrad, 1990), based on a set of consecutive bacteriological and serological studies, providing registration of a rise in antibody titer UPM (level of specific antibodies in RA autoslalom or TPHA with the erythrocyte diagnosticum), together with clinical symptoms, indicating about OKA the child, due to the UPM, and aimed to confirm the significance of isolated microorganisms and their associations.

However, the above method for the diagnosis of OKA mixed etiology concerns only bacterial-bacterial co-infections and does not affect the relationships in bacterial-protozoan associations, and is a very tedious and time-consuming diagnostic and not obespecivaet precision.

Currently available sources of method for the diagnosis of protozoan-bacterial OKA was not found.

Closest to the proposed method is a method of diagnosis of mixed forms described in MU No. 04-723/3 ot, "Guidelines for the microbiological diagnosis of disease caused by the enterobacteria", and MUK 4.2.735-99 "Parasitological methods for the laboratory diagnosis of helminthiasis and protozoosis", 2000, According to this method diagnosis of Salmonella infection is mainly through bacteriological studies using standard methods of isolation and identification of Salmonella. The research involves the isolation of Salmonella in the bacteriological laboratory. In this study faeces of the patient on intestinal protozoosis performed in the clinical laboratory according to the standard technique. Thus the main disadvantage of the above method is the research samples of faeces from patients with suspected Salmonella-protozoal OKA in different laboratories: biological - for allocation of Salmonella and clinical - carrying protozoology. In this study samples for Salmonella and protozoosis not always carried out from one portion of the faeces of the patient. This algorithm research does not provide evidence of the mixed nature of Salmonella-protozoal OKA, does not have sufficient efficiency and accuracy.

To eliminate the above disadvantages, the authors proposed a fundamentally new method of diagnostics is Tiki current Salmonella-acute intestinal protozoal infections.

The technical result is to increase the accuracy of diagnosis by quantitative evaluation of adhesive activity simultaneously analyzed samples of faeces as Salmonella and intestinal protozoa.

This result is achieved by the fact that in the known method, including the study of samples of faeces of patients with acute intestinal infections bacteriological method for Salmonella and microscopic method for protozoan cysts, according to the invention a sample of feces is concentrated and the obtained suspension is prepared 3 stroke, simultaneously within 1-1,5 h paint by carbolic fuchsin for Zn to indicate acid intestinal protozoa, 1% Lugol solution for the detection of Giardia, polyvalent adsorbed serum in the reaction of indirect immunofluorescence for indication of Salmonella and in the presence of specific glow of Salmonella on the surface of protozoa cysts establish a mixed etiology Salmonella-protozoal OKA, and the number of adhered cells of Salmonella on cysts in the field of view equal to from 1 to 4, diagnosed with a mild form, from 5-12 cells protracted form of infection.

Share discovery and evidence of etiological significance of protozoa cysts in Association with Salmonella using the method proposed by the authors, was 89%, whereas the detection of and the of solanto using the prototype method isolated detection bases on two different laboratories, in comparison with the author's method, amounted to only 15%(see drawing).

We first proposed the simultaneous preparation of smears for staining as the acid intestinal protozoa, Giardia, and Salmonella.

We offer pre-concentration and enrichment of feces samples by ether-acetic sedimentation protozoa cysts. In addition, we found the maximum concentration of cysts needed for further study samples for Salmonella.

For the first time, we have established the ability of Salmonella to show adhesive activity to the surface of protozoa cysts, which is the main mechanism for the development of infectious process, similar to the mechanism of adhesion of cells navigatethrough epithelium, as well as free cells of multicellular organisms. The authors found that Salmonella, adhered on the surface of protozoa cysts, is evidence of mixed infections with Salmonella-protozoal process in the intestine, since the stages of dyeing and washing when setting reacii indirect immunofluorescence completely eliminates the possibility of non-specific nature of the adhesion of Salmonella on the cysts.

The difference of this method is the fact that the diagnosis of salmonellosis is a protozoal infection ZAT is aceveda 1-1,5 h, unlike most similar prototype method in which the indication of Salmonella and intestinal protozoa requires 24-48 hours. We for painting using Salmonella adsorbed multivalent D serum, confirming the specificity of fluorescence in the reaction of indirect immunofluorescence on Salmonella.

The authors first propose a quantitative account of adhered on the cysts of intestinal protozoa of Salmonella and the number of adhered cells of Salmonella on cysts in the field of view equal to from 1 to 4, diagnosed with mild, from 5-12 cells of Salmonella - severe flow mixed infection, thereby predicting the severity of prolonged Salmonella-protozoal OKA.

The proposed method is as follows. Examinations of stool samples from patients with acute intestinal infections Salmonella etiology, transported to the laboratory, are ether-acetic sedimentation to achieve the greatest concentrations potentially present in them cysts and trophozoites of intestinal protozoa. From the resulting precipitate simultaneously prepare three strokes. In one medication immediately make a drop of 1%aqueous solution of Lugol's solution for the differential diagnosis of amoebae and flagellates protozoa, including Giardia and dientamoeba. Mikroskopiruyut, noting adhesion on the surface of the CIS is small sticks.

Two other drugs dried at room temperature, then fixed in cold acetone and stained by carbolic fuchsin for Zn Nillsen the detection of oocysts of cryptosporidia and isospora. In case of detection of oocysts, painted in red, say adhesion on the surface of small, painted in green sticks. The remaining product is subjected to investigation in the reaction of indirect immunofluorescence with the use of diagnostic adsorbed Salmonella polyvalent O-whey, major groups a, b, C, D, E. a fixed stroke put a drop of adsorbed rabbit polyvalent Salmonella O-serum a, b, C, D, E. Then the product is embellished individuai luminescense serum with simultaneous contrast. The specificity of adhesion confirmed by carrying out stages of dyeing and washing when setting reaction of indirect immunofluorescence and completely eliminates the possibility of non-specific nature of the adhesion of Salmonella on the cysts.

A positive result is considered to be specific bright luminescence (fluorescence) greenish light on the periphery of the cysts (in the area of adhesion of Salmonella on the surface), clearly contrasting with the dark body of cysts of intestinal protozoa. The morphology of the species, as a rule, is stored. Evaluation of the drug should carry out what I comprehensively taking into account such factors as brightness and color specific fluorescence, localization and structure of the glow, and the number of adhered on the cysts of protozoa of Salmonella in the field of view.

To establish the form of the disease is the average of adhered on the surface of the cyst cells of Salmonella in the field of view. For this purpose, microscopic examination under a fluorescent microscope, looking at at least 3 fields of view, each of which produce the counting of cysts and adesione of Salmonella. Then calculate the average value of adhered cells. If the value is equal to from 1 to 4, diagnosed with a mild form, from 5-12 cells of Salmonella - long form.

For a full range of studies by the author's method and diagnosis of salmonellosis is a protozoal infection takes 1-1,5 h, in contrast to the closest prototype of the way in which the indication of Salmonella and intestinal protozoa requires 24-48 hours.

Thus, the advantage of the proposed method for the diagnosis of the current Salmonella-acute intestinal protozoal infections in children is its high sensitivity and specificity, simplicity, availability, accuracy of diagnosis, the possibility of obtaining results through 1-1,5 hours after sampling of the material, and the use of the method in the early diagnosis of clinical f the RM Salmonella is a protozoal infection, and surveys of patients with Salmonella hosting.

The inventive method can be confirmed by the following examples.

Example 1.

Patient C., 5 years 2 months. The diagnosis of salmonellosis typhimurium var. Copenhagen, severe form, acute.

OptionsIndicators prior to the survey according to the method of diagnosis of the nature of the flow Salmonella is a protozoal infection in childrenIndicators of the change of tactics of treatment after the application of the method of diagnosis of the nature of the flow Salmonella is a protozoal infection in children
1. The terms of admission to the hospital7 day diseases-
2. Bed-days20 days-
3. Smear-positive28 days
4. DiagnosisAdmission: salmonellosis of typhimurium var. Copenhagen, severe form, acuteFinal: mixed intestinal infection. Salmonellosis. Giardiasis. Gus is trointestinal protracted form. Acute course. Severe
4 the Presence of JITSymptoms GIT and changes of the gallbladder and bile ducts on ultrasoundSymptoms GIT with less intensity and frequency, the disappearance of the changes on ultrasound
5. The clinical analysis of bloodEosinophils 12-14Eosinophils 1-2
ESR up to 34 mm/hErythrocyte sedimentation rate of 4 mm/hour
Hb increased to 135 g/lHb 110 g/l
Leukocytosis up to 9Leukocytes 2
Monocytes to 7-9Monocytes 1-2
6. CoprogramFATS:FATS:
neutron. fats up to 3neutron. fats 1
washed. sour. to 2washed. sour. to 2
soap to 2soap 1
A large amount of mucusLeukocytes 2-3
Leukocytes cover vs the field of view (50-60), neutrophilic in natureCell prismatic epithelium in a/C no
Cell prismatic epithelium in the p/C
Ons symptoms: pain in the course of bowels, fever, symptoms of intoxication, irregular bowel movements, vomiting+-
8. The performance of the method for the diagnosis of the nature of the flow Salmonella is a protozoal infection in childrenMassive colonization cysts of GiardiaNo smears cysts with adhered on them by Salmonella
The average value of adhered on the surface of cysts in P.Z. 10

As can be seen from the data, after the author's technique changes were made to the wording of the diagnosis of mixed salmonellosis giardiasis infection was modified strategy etiotropic therapy that determined the normalization of laboratory parameters and clinical recovery of the patient.

Example 2.

Patient K., 2 months. The diagnosis of salmonellosis typhimurium, moderate flow, gastrointestinally form.

OptionsIndicators prior to the survey according to the method of diagnosis of the nature of the flow Salmonella is a protozoal infection in childrenIndicators of the change of tactics of treatment after the application of the method of diagnosis of the nature of the flow Salmonella is a protozoal infection in children
1. The terms of admission to the hospital9 day diseases-
2. Bed-days19 days-
3. Smear-positive20 days-
4. DiagnosisAdmission: salmonellosis of typhimurium, moderate flow, gastrointestinally formFinal: mixed intestinal infection. Salmonellosis. Giardiasis. The gastrointestinal form. Acute course. Severe
4 the Presence of JITSymptoms GIT and changes of the gallbladder and bile ducts on ultrasoundSymptoms GIT with less intensity and frequency, the disappearance of the changes on ultrasound
5. The clinical analysis of bloodEosinophils 3Eosinophils 1-2
ESR up to 32 mm/hourErythrocyte sedimentation rate of 4 mm/hour
Hb increased to 94 g/lHb 105 g/l
Leukocytosis up to 10-13Leukocytes 2
Monocytes to 5Monocytes 1-2
6. CoprogramFATS:FATS:
neutron. fats up to 3neutron. fats 1
washed. sour. to 2washed. sour. to 2
soap to 2soap 1
A large amount of mucusLeukocytes 2-3
Leukocytes cover the entire field of view (50-60), neutrophilic in natureCell prismatic epithelium in a/C no
Cell prismatic epithelium in the p/C
7. Clinical manifestations: pain during bowel, fever, symptoms online is Xiali, irregular bowel movements, vomiting+-
8. The performance of the method for the diagnosis of the nature of the flow Salmonella-protozool infection in childrenMassive colonization cysts of GiardiaNo smears cysts with adhered on them by Salmonella
- The average value of adhered on the surface of cysts in PS

As can be seen from the above data, clinical diagnostic indicators in the group of children with Salmonella-mixed protozoal infection are expressed in the distortion typical clinical manifestations as salmonellosis and giardiasis infections manifesting a new infectious disease, and are characterized by longer and wavy course of the OKA, after setting of the author's methodology changes were made to the wording of the diagnosis of mixed salmonellosis giardiasis infection was modified strategy etiotropic therapy that determined the normalization of laboratory parameters and clinical recovery of the patient.

The proposed method is tested on the material (samples of faeces) from 110 patients OKA (salmonellosis). The results of the study of the nature of the flow Salmonella-protozoal m of the CRP-infection with a quantitative assessment of cells of Salmonella, adhered on the surface of protozoa cysts, has allowed to establish that when the number of adhered on the cysts of Salmonella equal to 1-4, there is an easy degree, and when the amount of from 5 to 12 severe currents Salmonella-mixed protozoal infection that was Koroliova with the severity of the condition and the duration of stay of children in hospital. The clinical picture in patients with mixed protozoal infection in contrast to patients with monoclonality was characterized by:

1) later maturity in the hospital, the increase in bed-days;

2) increase the period of Salmonella bacteria, often rolling in hosting;

3) admission to the hospital on a background of reception of the current comorbidities;

4) symptoms GIT and specific changes in the gall bladder and liver on ultrasound;

5) allergic reactions in the anamnesis;

6) longer feverish period subfebrility numerals;

7) more pronounced syndrome exitosa with toxemia (I, II, art);

8), increasing frequency of vomiting, for a longer period of their registration;

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

10) more pronounced qualitative and quantitative what izmeneniami when intestinal dysbiosis;

11) specific changes in the results.

Thus, clinical and diagnostic features in the group of children with Salmonella-protozoal mixed infections are expressed in the distortion typical clinical manifestations as Salmonella and protozoan infections, manifesting a new infectious disease, and are characterized by longer and wavy course of the OKA.

The method, developed by the authors, can be recommended for widespread use in medical practice for the diagnosis flow (mild, severe) Salmonella-protozoal acute intestinal infections (AII) in children. The method has high sensitivity and specificity, simplicity and accessibility. The possibility of obtaining results through 1-1,5 hours after sampling of the material, and the use of the method in the early diagnosis of clinical forms of salmonellosis is a protozoal infection, and in the examination of patients with Salmonella hosting, determines its enormous practical importance.

The way to diagnose the current Salmonella-protozoal infections in children by examining samples of faeces of patients with acute intestinal infections of bacterial and microscopic method, wherein the pre-one sample of faeces pain the CSOs concentrate by ether-acetic sedimentation, from the resulting sludge is prepared simultaneously three stroke: first, paint the carbolic fuchsin for Zn to indicate acid intestinal protozoa, second - 1% Lugol solution for the detection of Giardia, third - polyvalent adsorbed serum in the reaction indirect immunofluorescence assay for indication of Salmonella and when it detects a specific glow diagnose Salmonella-acute intestinal protozoal infection, and if their number is in the field of view is 1-4 cells diagnosed with a mild form, from 5-12 cells - severe form of the infection.



 

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1 ex, 2 tbl

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5 cl, 1 ex, 4 tbl

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5 tbl, 2 dwg

FIELD: medicine.

SUBSTANCE: zeolite antioxidant activity test is enabled by introducing a substance being tested into bodies of experimental animals. Biological products of tissues and organs of the experimental animals and control sets are prepared. Metabolic process indicator substances are evaluated. The pulmonary tissue, blood plasma, erythrocytes and thrombocytes are analysed for the content of lipid peroxidation products and natural antioxidants which are scored and summed up. The zeolite antioxidant activity is tested relatively to the normal values of the content of lipid peroxidation products and natural antioxidants which are defined as an arithmetical mean of the relevant values received in the animals of a control set.

EFFECT: enabled reliable zeolite antioxidant activity test at the enabled comparative evaluation of substances by this parametre.

2 tbl, 1 ex

FIELD: medicine, hepatology.

SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.

EFFECT: higher accuracy of diagnostics.

3 ex

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