A method for the diagnosis of cryptosporidiosis


G01N1/30 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

 

The invention relates to the field of medicine. A method for the diagnosis of cryptosporidiosis include histological examination of biopsy, the final fixation of the sections of methanol exposure at colouring by carbolic fuchsin is carried out for 5-10 minutes, as decolorizer apply 5% sulfuric acid. The method provides a positive effect in the form of identifying objective criteria for the diagnosis of cryptosporidiosis, providing treatment. 1 table, 3 Il.

The invention relates to medicine, namely to Microbiology, and can be used in complex laboratory diagnosis of gastrointestinal diseases.

Cryptosporidium are obligate parasites infecting microforming mucous membranes of the digestive tract and the respiratory tract. Infections caused by cryptosporidia, rarely diagnosed. The under-reading of cryptosporidiosis associated with imperfect diagnostic technologies.

A known method for the diagnosis of cryptosporidiosis using microscopic examination of a smear of faeces (“Clinical Microbiology and antimicrobial chemotherapy”, No. 3, volume 2, 2000, S. 51-57), including its gram stain. However, as the criminal code of jiaodong mushrooms.

There is a method of determining the discharge of oocysts of cryptosporidia using the staining method of the Zn (Merkulov, A. Course patrologicheskoe technology. - L.: Medicine, 1969, S. 301).

However, this method allows when using the microscope to determine only the oocyst of Cryptosporidium in the population. The accuracy varies from 2% to 6%, but the average does not exceed 4%.

For the prototype of the present invention, the selected method of identifying cryptosporidia in paraffin tissue sections colouring on Zn (Zinserling A. C. Processing and staining of smears and sections for the detection of microorganisms //Archives of pathology, No. 5, volume 54, 1992, S. 36), including morphological identification of oocysts of cryptosporidia in paraffin sections.

The method is as follows:

1. The slice is covered with filter paper, the paper is poured a solution of carbolic fuchsin (1 part saturated solution of basic fuchsin in 10 parts of 5% aqueous phenol), then the slice is heated until the vapor, leaving the paint on the cut for another 20-30 minutes, or without heating up leave in the dye for 15-18 hours

2. Taking the paper, rinse slice in water.

3. Differentiate in 1% hydrochloric acid alcohol to pale pink.

4. Washed in water.

5. Paint GE is the way oocysts of cryptosporidia crimson color revealed on the background fabric, stained with hematoxylin or methylene blue. A significant drawback of the method is that only identified oocysts, and the intermediate stage is not detected, which reduces the diagnostic accuracy of 66%.

The aim of the invention is to improve the accuracy of the method and advanced diagnostic capabilities.

This objective is achieved in that in the method for the diagnosis of cryptosporidiosis by histological examination of biopsy is conducted additionally the final fixation of the sections of methanol. Exposure at colouring by carbolic fuchsin is carried out for from 1 to 5 minutes, as decolorizer apply 5% sulfuric acid. The results of the study are presented in Fig.1, 2, 3.

In Fig.1 presents the mucous membrane of the colon of the patient E., age 9: the oocysts in the overlay, the surface epithelium of the zone of lysis and its adhesion to the epithelium of the crypts. The colouring on Zn400 (example 1).

In Fig.2 presents the mucous membrane of the antrum of the stomach of the patient C., 14: b - adhesion sporozoite on the surface epithelium, in - meranti I type on the surface epithelium. Coloring basic fuchsin in Zn400 (example 2).

petelia intestinal villi. Coloring basic fuchsin in Zn400 (example 3).

The method is as follows:

1. Spend the final fixation dewaxed sections in absolute methanol for 5-10 minutes

2. Dried out.

3. Paint a saturated solution of basic fuchsin for 5 minutes

4. It is dried over a flame.

5. Incubated in 5% sulfuric acid solution for 1 min.

6. Rinse in 70% alcohol prior to bleaching (5-10 s).

7. Domracheva aqueous solution of methylene blue from 10 sec to 1 min.

8. Rinse in water.

9. Sign in canadian balsam.

The rationale for the health of this method is the following: the use of dofixture methanol increases electively to dye thin-walled oocysts formed oocysts and microgame, and the use of 5% sulfuric acid as decolorizer contributes to the binding of the dye.

Examples of specific performance are given in the form of extracts from the histories.

Example No. 1.

Sick of Sinohub Eugene, 9 years old. Clinical diagnosis: hemocrit of unclear Genesis.

History: the Child from first pregnancy, which occurred at the background of toxicity. On breastfed up to 3 months.

Data instrumental methods:

Irigaray intestine - the large intestine is usually moderately elongated in the sigmoid colon.

Esophagogastroduodenoscopy - pathology it is not revealed.

Colonopenbracket endoscope is introduced in the ileal mucosa (WITH) her pink, velvety with a single follicles, ileocecal valve gubbini forms, closed, oriented in the ascending colon. WITH the colon with faint spotting, edema, vascular pattern is irregular, blurred in many areas. The folds of the usual dimensions, ground clearance is not deformed. Executed floor biopsies FROM the ileum, colon and rectum.

Conclusion - colitis.

When using the microscope sections of the mucous membrane of the colon is determined by the oocysts in the overlays on the surface of the epithelium with a zone of lysis and adhesion to the epithelium of the crypts. The colouring on Zn400 (Fig.1).

Treatment: prescribed diet with the exception of lactose and fat, rehydration therapy polyion is 3 times a day for 2 weeks. After the treatment chair is normalized. Discharged home in good condition.

Example No. 2.

Patient Itskov Sasha, 14 years. Was in the hospital with a clinical diagnosis of chronic gastroduodenitis in the acute stage.

During esophagogastroduodenoscopy showed signs of terminal esophagitis, chronic gastroduodenitis. Biopsies. Morphological conclusion: chronic diffuse antral gastritis, acute exacerbation. The drug is painted the proposed method: when using the microscope sections of the mucosa of the antrum is determined by the adhesion of sporozoite on the surface of the epithelium and Maranta I type on the surface of the epithelium. Coloring basic fuchsin in Zn400 (Fig.2).

After carrying out of a course of treatment trichopol discharged healthy.

Example No. 3.

Patient Ivanova Ekaterina - 6 years. Clinical diagnosis: chronic gastroduodenitis in the acute stage.

During esophagogastroduodenoscopy revealed signs of chronic gastroduodenitis. Biopsies. Morphological conclusion, chronic diffuse antral gastritis, duodenitis - aggravation. When using the microscope slices. basic fuchsin for Zn Nilsso400 (Fig.3).

Treated with trichopol in 2 weeks. Discharged after three weeks in a satisfactory condition.

This method examined in 43 children (clinical diagnosis: diarrhea syndrome of unknown etiology, hemocrit unclear etiology, chronic recurrent gastro) - total drugs 71 (table).

Search etiological mechanisms showed that 25 patients a cause of diarrhea is the presence of Cryptosporidium in the mucosal obolochke particular digestive tract. Cryptosporidium was detected in 50% of patients in the studied sections of the gastric mucosa, 50% of the small intestine, 39% of colon. The accuracy of the method is 45%.

Advantages in comparison with known:

1. Raising awareness and accuracy, because it allows to obtain integrative assessment cryptosporidial invasion of the mucous membrane of any of the digestive tract.

2. The method is reliable in performance, as the final fixation in methanol and differentiation in a 5% solution of sulfuric acid allows to detect Cryptosporidium at all stages of development - from Maranta to oocysts.

3. The method does not require expensive equipment, use imperatoria medical institutions.

4. How has the economic efficiency so as to reduce the period of analysis simultaneously considers the structure of the tissue, which is 2 times reduces the consumption of working time, dyes, slide, slide cover glasses.

The method can be carried out directly on this description.

Claims

A method for the diagnosis of cryptosporidiosis, including histological examination of biopsies from the gastric mucosa, 12 duodenal, jejunal, ileal and colonic gut and fixation in 10% neutral buffered formalin, characterized in that it further conduct the final fixation of the sections of methanol exposure at colouring by carbolic fuchsin is carried out for 5-10 minutes, as decolorizer apply 5% sulfuric acid.

 

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1 cl, 1 dwg

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