RU2638810C1 - Method for determination of lamblia cysts and cryptosporidia oocysts in clinical material, washouts from environmental objects, in soil - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is a method for lamblia cysts and cryptosporidia oocysts determination in one formulation in biological material - feces, in the washouts of environmental objects and soil, including sample preparation, immunomagnetic particles introduction into the sample, immunochemical binding, resulting in cyst and oocyst aggregation with magnetic particles, capture of cyst and oocyst aggregates and capture in the magnetic field, fixed cyst and oocyst aggregates by buffer solution, dissociation with mercaptoethanol or hydrochloric acid, cyst, oocyst and magnetic particles separation in the magnetic field, transfer of selected cysts and oocysts to a slide for subsequent immunofluorescent marking and subsequent microscopic evaluation using the "Opti-Lum" cap for the microscope, where the result is taken into account assuming that lamblia cysts are sparkling and apple-green fluorescent objects, from rounded to oval, 8 to 14 microns in length, 7-10 mcm in width, with brightly lit edges, while cryptosporidia oocysts represent sparkling and apple-green fluorescent objects, from oval to spherical, 3 to 5 mcm in diameter, with brightly lit edges.

EFFECT: improved effectiveness of lamblia cysts and cryptosporidia oocysts determination in one formulation in biological material - feces, in the washouts from vegetables and environmental objects, in soil, and reduced time expenses for study of one sample of investigated material adapted to 15 minutes.

7 ex, 7 tbl, 1 dwg

Description

The present invention relates to the field of clinical medicine, more specifically to work on the determination of parasitological agents in the clinical material - feces, in washes of environmental objects, in the soil by the adapted method of immunomagnetic separation followed by immunofluorescence labeling with the developed sample preparation of biological material for research.

The current stage of development of parasitological laboratory studies allows a comprehensive assessment of the parasitic system using microscopic, immune, immunochemical and high-tech analysis methods, including polymerase chain reaction method and the use of a complex of automated microscopy. In recent years, the methodology of parasitological analyzes has undergone radical modernization, which made it possible to create a modern level of the doctor's working conditions, accuracy and productivity. However, in the analytical processing of indicators of the parasitic morbidity of the population, independent experts use the terms “true” and “recorded” prevalence of parasitoses, emphasizing the low efficiency of the diagnostic work of health facilities and inappropriate statistical indicators of environmental pollution by parasitic pathogens. To date, in parasitological laboratories, when determining the quality of water for the presence of cryptosporidium oocysts and lamblia cysts, archaic classical methods are used with very low sensitivity.

A known method for the study of water for the presence of lambli cysts and cryptosporidium oocysts by the method of immunomagnetic separation and labeling with fluorescent antibodies, including sampling, conducting studies and evaluating the results. (Methodical instructions MUK 4.2.2314-08, MUK 4.2.2959-11). The indicated method is quite long in time and laborious and requires expensive equipment and reagents for its implementation, limited to the object of study - water.

The technical result achieved by using the invention is to increase the efficiency of the complex determination of helminth eggs, cysts of pathogenic intestinal protozoa, including cryptosporidium oocysts in one formulation by immunomagnetic separation followed by immunofluorescence labeling in biological material of feces, in washes of environmental objects, in soil and reduction time spent on the study of one sample of the test material up to 15 minutes.

The technical result is achieved by the fact that in a method for determining in one formulation cystosporidium lamblia and oocysts in biological material - feces, in washes of environmental objects, in soil, including sampling, conducting research and evaluating the results, when conducting studies when binding and washing cysts Giardia and cryptosporidium oocysts transfer the resuspended test sediment to a magnetic tube and evaluate by microscopy using an Opti-Lum nozzle on a microscope, the result is taken into account Since lamblia cysts are sparkling and fluorescent apple-green objects, from round to oval from 8 to 14 microns in length 7-10 microns wide, with brightly lit edges, cryptosporidium oocysts are sparkling and fluorescent apple -green light objects, from oval to spherical from 3 to 5 microns in diameter, with brightly lit edges.

The proposed method is implemented as follows.

The procedure for binding and washing cystosporidium lamblia and oocysts is as follows: add 5 ml of the buffer from the diagnostic kit to a magnetic tube with a flat side surface for immunomagnetic separation, close the tube with a cap and rotate the tube, wetting the inner walls of the buffer; a diagnostic residue is introduced into it. The entire volume of the immunomagnetic suspension of the Cryptosporidium Beads and Giardia Beads diagnostic kit is vortexed for 10-15 seconds, 100 μl (0.1 ml) of the suspension is taken from each vial with a microdoser and transferred to an IC tube, which already contains a portion of diagnostic sediment. Close the lid, fix the magnetic tube in a laboratory rotator rack and mix for one hour at a speed of 15-25 rpm with swinging movements (without turning the tube over!). Then the magnetic tube is removed from the rotator and placed in a MagnetOn 4T ™ magnetic tripod; with the flat side of the tube facing the tripod magnet; for 2 min, without turning the tube over, tilt the tube with the magnetic stand in the direction from the bottom to the tube cover and vice versa. As you move on the flat side of the magnetic tube, a precipitate will form. Without removing the IC tube from the magnetic tripod, open the lid and carefully drain the supernatant from the tube. Add 1 ml of IT-Wash Buffer (WB) washing buffer to a magnetic tube, remove the tube from a tripod, and shake it to completely flush the sediment from the flat side of the tube. Using a SepPen magnetic pen, magnetic suspension is collected. SepPen magnetic pen is removed from the magnetic tube.

In an Epindorf type microcentrifuge tube (1.5-2 ml in volume), 1 ml of WB washing buffer is placed and the previously collected suspension is introduced into it. 1 ml of WB buffer is added to the second magnetic tube, the lid is closed and the inner walls of the tube and lid are washed with a buffer, the magnetic tube is placed in a Magnet On4T ™ magnetic stand; tilting right and left for 30-60 s, wash the inner surface of the tube and collect the remnants of the magnetic suspension. Without removing the magnetic tube from the magnetic tripod, WB washing buffer is drained from the tube and a new 1 ml portion of WB washing buffer is added to the tube. The lid is closed and the washing procedure is repeated. Next, the magnetic tube is removed from the tripod and, with agitation, the suspension is washed off from the flat side of the tube. Using a SepPen magnetic pen, the resulting suspension is transferred to a microcentrifuge tube, into which WB washing buffer and the first portion of the processed diagnostic sediment were previously added, placed in a magnetic tripod and tilted left and right for 1 min, left for 15 s in a vertical position. Without removing the microcentrifuge tube from the magnetic tripod, the WB washing buffer is drained from the tube, 1 ml of a new portion of the WB washing buffer is added to the tube.

The dissociation procedure is as follows: without removing the microcentrifuge tube from the magnetic tripod, open the lid and drain the washing buffer from the tube, add 100 μl of working solution of 2-mercaptoethanol to the tube, mix the suspension on a vortex for 30 s and place in a thermostat or water bath at 50 ° C for 5 min, then re-suspension in a microcentrifuge tube was vortexed for 30 s and mounted in a magnetic tripod. Without removing the tube from the magnetic tripod, the supernatant is removed and transferred to the SuperStick ™ slide for subsequent immunofluorescence labeling. A drop of positive control, G. lamblia, C. parvum, is introduced into the second window of slide S100-2.

The detection (identification) of cryptosporidium and giardia cysts by immunofluorescence labeling is carried out in several stages. Preparation for tagging. One drop (about 45 μl) of the working solution of the AquaGlo ™ G / C * immunoreagent is added to each well of the slide with a dried sample and control. Slides are placed in a humidity cell in the dark and incubated for at least 30 minutes at 37 ° C or at least 40 minutes at room temperature. One drop of phosphate buffer (PBS) or saline is applied to each well and incubated for at least 2 minutes. Each slide is tilted onto clean filter paper and the excess liquid is carefully aspirated.

The labeling procedure is as follows: one drop of contrasting dye is added to each well and incubated for 1 min at room temperature, then one drop of phosphate buffer (PBS) diluted with distilled water (1:19) or saline washing buffer is added to it. 20XWash Buffer and incubated for 1 min at room temperature. Excess fluid from the bottom of the slide wells is aspirated with filter paper or a Pasteur pipette. The slides are laid out on an inclined tripod and dried at room temperature. Then, 1 drop of No-Fade ™ mounting medium is added to the wells, covered with a coverslip, and the edges are sealed with Canadian balsam or colorless varnish.

The preparations are microscopic under oil immersion using a luminescent microscope or using the Opti-Lum nozzle, which converts a light microscope into a luminescent one. Investigate drugs at x200. Before starting microscopy of diagnostic preparations, the positive control preparations from the diagnostic set with counted Giardia cysts and cryptosporidium oocysts are preliminarily examined.

The result is taken into account on the basis that lamblia cysts are objects that sparkle and fluoresce with apple-green light, from round to oval (8-14 microns in length by 7-10 microns in width), with brightly highlighted edges; cryptosporidium oocysts are objects that sparkle and fluoresce with apple-green light, from oval to spherical (from 3 to 5 microns in diameter), with brightly illuminated edges.

The proposed method is illustrated by examples.

Example 1. Identification of a number of advantages of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies in a comparative assessment of classical methods for detecting parasitological pathogens from various environmental objects and clinical material (Table 1).

The adapted method of immunomagnetic separation and labeling with fluorescent antibodies takes 25 minutes for sample preparation, in addition to etherformalin, it allows you to quickly take the result into account, the preparations are stored for several months, cryptosporidium oocysts are determined only by the IC, except for the additional Ziehl-Nielson staining method in the study of clinical material - feces, 100 % the reliability of the result on the detection of lamblia cysts and cryptosporidium oocysts (table 1).

In FIG. 1. Schematically depicts the steps of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies.

Example 2. An experimental series of studies was conducted to determine the sensitivity of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies for all types of samples. As a result, it was found that the sensitivity of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies when studying samples of environmental objects without loss of the studied material at the stage of sample preparation (feces, washings from surfaces) is up to 100%. The sensitivity of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies in the study of samples with a partial loss of the studied material at the stage of sample preparation (soil) is up to 85-90% (Table 2).

Example 3. In the study of 650 samples of full-scale material by 2 methods, 100% efficiency of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies in determining cystosporidium lamblia and oocysts was established. To determine the specificity of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies, experimental models with artificial infection of field material were used. The efficiency of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies in the experimental series of stool samples studies was 100% lamblia cysts, 100% cryptosporidium oocysts with experimental formaldehyde sediment studies 95.0% lamblia cysts respectively, 0 - cryptosporidium oocysts, table 3 (not found )

Example 4. When examining 900 samples of field material by 2 methods, sediment and supernatant, 90.0% detectability of lamblia cysts and 70.0% cryptosporidium oocysts by the adapted method of immunomagnetic separation and labeling with fluorescent antibodies was established, which is explained by a partial loss of parasitic pathogens during sample preparation.

The detection detectability is about 30.0% of lamblia cysts and 0% of cryptosporidium oocysts using the Padchenko method. With the efficiency of determining parasitic pathogens in the soil, the adapted method of immunomagnetic separation and labeling with fluorescent antibodies determines 90% of lamblia cysts and 100% of cryptosporidium oocysts. To determine the specificity of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies, experimental models with artificial infection of field material were used. The efficiency of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies in the experimental series of studies was 100.0% lamblia cysts, 100.0% cryptosporidium oocysts, unlike the Padchenko method, whose efficiency was 55.0% for lamblia cysts, 0% for cryptosporidium oocysts (Table 4).

Example 5. In the study of 900 samples of full-scale sediment material and supernatant by 2 methods, 90.0% detectability of lamblia cysts and 70.0% cryptosporidium oocysts was determined using the adapted method of immunomagnetic separation and labeling with fluorescent antibodies, due to the partial loss of parasitic pathogens during sample preparation.

By the Padchenko method, about 30.0% of lamblia cysts and 0% are cryptosporidium oocysts. The percentage of the effectiveness of the determination of parasitic pathogens in soil by IMS is 90% of Giardia cysts and 100% of cryptosporidium oocysts.

To determine the specificity of the adapted method of immunomagnetic separation and labeling with fluorescent antibodies, field studies of 20 flush samples from the surfaces of environmental objects (samples were taken in swimming pools for children and adults, objects - floors, walls, door handles) and experimental models with artificial infection of flush samples were used. According to the results of surface washings using an adapted method of immunomagnetic separation and labeling with fluorescent antibodies, 5 lamblia cysts and 3 cryptosporidium oocysts were detected, and lamblia cysts and cryptosporidium oocysts were not detected by the Romanenko method. Samples of washes from the surfaces of the pools artificially infected 20 units. Giardia cyst and 20 units. cryptosporidium oocyst. The efficiency of the determination of lamblia cysts and cryptosporidium oocysts in the experimental series of studies by the adapted method of immunomagnetic separation and labeling with fluorescent antibodies was 100.0%, in contrast to the Romanenko method, whose efficiency was 55.0% of lamblia cysts and 0% cryptosporidium oocysts (Table 5).

Example 6. The study of 500 samples of field material from fruit and vegetable products (beets, carrots, potatoes, strawberries) was carried out by 3 methods: the supernatant was studied by the Romanenko method and the adapted method of immunomagnetic separation and labeling with fluorescent antibodies, the soil sediment was studied by sequential filtration through analytical track filtration membranes and an adapted method of immunomagnetic separation and labeling with fluorescent antibodies. In the supernatant, 100% efficiency was determined for the determination of lamblia crypts and cryptosporidium oocysts by the adapted method of immunomagnetic separation and labeling with fluorescent antibodies. The percentage of detection of parasitic pathogens in the study of soil sediment by the classical filtration method is 25.0% for cystosporidium cystic sporidia and 0 cryptosporidium oocysts, and 90.0% for cryptosporidium cystosporidia using immunomagnetic separation and labeling with fluorescent antibodies (Table 6).

Example 7. Comparative evaluation of classical methods for detecting parasitological pathogens from various environmental objects and in clinical material in comparison with the adapted method of immunomagnetic separation and labeling with fluorescent antibodies and identifying a number of advantages of the proposed method: it takes 25 min for sample preparation; allows you to quickly take into account the result; drugs are stored for several months; cryptosporidium oocysts are determined only by the adapted method of immunomagnetic separation and labeling with fluorescent antibodies, except for the additional Ziehl-Nielson staining method in the study of clinical material (feces); 100% reliability of the result on the detection of lamblia cysts and cryptosporidium oocysts (table 7).

Thus, the high sensitivity, specificity and target orientation of the method for determining cystosporidia lamblia cysts and oocysts in the clinical material - feces, in washes from environmental objects and in the soil by the adapted method of immunomagnetic separation followed by immunofluorescence labeling allows us to determine small doses of parasitic pathogens in the environment, which is especially important in the sanitary-epidemiological and environmental risk assessment in areas with a characteristic for the area and / or at a pathogen remote from the source of circulation. The indicated characteristics of the new method, the possibility of its application for a comprehensive assessment of the parasitological situation, will ensure the reliability of studies, their information content and efficiency in making managerial decisions and help increase the effectiveness of state monitoring in the field of health and the environment.

Claims (1)

A method for determining in one formulation lamblia cysts and cryptosporidium oocysts in biological material - feces, in washes of environmental objects and in soil, which consists in preparing a sample, introducing immunomagnetic particles into the sample, immunochemical binding, as a result of which aggregates of cysts and oocysts with magnetic particles are formed trapping aggregates of cysts and oocysts in a magnetic field, washing the fixed aggregates of cysts and oocysts with a buffer solution, dissociation with mercaptoethanol or hydrochloric acid, separation of cysts, oocysts and a magnet particles in a magnetic field, transferring the selected cysts and oocysts to a glass slide for subsequent immunofluorescence labeling and subsequent microscopic evaluation using an Opti-Lum nozzle under a microscope, where the result is taken into account based on the fact that lamblia cysts are sparkling and fluorescent apple green light objects from rounded to oval from 8 to 14 microns in length 7-10 microns wide, with brightly lit edges, the cryptosporidium oocysts are sparkling and fluorescent apple-green with objects that are oval to spherical from 3 to 5 microns in diameter, with brightly lit edges.

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