Method for detection of helminthes, tick eggs and protozoan oocysts in soil samples

FIELD: medicine.

SUBSTANCE: method for detection of helminthes, tick eggs and protozoan oocysts in soil samples consisting in the fact that a soil sample 25 g is placed in a centrifugal test-tube 250 ml and filled with water 150 ml; the mixture is thoroughly mixed with a glass rod for 5 min or with an electrical mixer for 1 min and centrifuged for 3 min at 800-1000 rpm; water is poured off, while the test tubes are added with a three-component flotation system 150 ml containing saturated solutions of zinc chloride (2 kg of ZnCl2 or 1 l), sodium chloride (0.42 kg of NaCl per 1 l) and glycerol taken in proportions 1:1:1; the test tube is added with the same solution to form a convex meniscus, covered with a degreased glass so that it touches a fluid layer; 20-30 min. later, the glass is removed and observed under microscope; the preparation is recommended to be drawn for 2-3 times. The detectability of protozoan oocysts and tick eggs increases by 76.6 and 87.1% respectively.

EFFECT: method enables considerably increased a percentage of detected eggs of such soil-transmitted helminthes as human and swine ascarid, twice reducing time for expecting the analysis results.

4 tbl, 2 ex

 

This invention relates to the field of medicine, veterinary medicine, ecology. Can be used for the detection of helminth eggs, ticks and oocysts in soil samples.

The most relevant assessment of contamination of soil eggs are often found in the sand, in particular on the playgrounds and sandboxes - Toxocara canis, Toxocara cati and Ascaris lumbricoides. To exercise the poultry yards of the simplest often found Amerie (Eimeria tenella, E. maxima, E. acervulina), and mites Knemidocoptes mutans.

The most frequently used method of soil testing to identify helminth eggs is combined method Romanenko N. A. where the flotation fluid saturated solution of sodium nitrate (MUK 4. 2. 796 - 99. Methods of sanitary Parasitology research").

The soil selected for the study on private and public gardens, fields, irrigation, public houses, kindergartens, markets, places for children to play, farms, places of residence of owners of cattle, etc. also Explore activated sludge and sludge treatment plants, sediments at the confluence of sewage or other effluents into water bodies. After examination of the site plan designated for sampling, where one can expect the presence of helminth eggs.

Soil samples taken from the surface and at a depth of 10-20 cm, and sludge drying beds, composting on the depth of -2 m

On the same site (the"inspection point") soil collected in 5-10 different places 10-20 g, combining samples in one total mass not less than 100-200, On the territory of one object (the estate, kindergarten, etc.) select 10 samples.

The sample is placed in a tightly closed glass container or plastic bag. Inside put a label indicating the place and conditions of sampling.

Samples delivered to the laboratory, it is desirable to investigate immediately, as the eggs of a number of worms or larvae may die and disintegrate. Store samples recommended in the refrigerator (5°C) not more than one month.

For the research method Naamanka of the total samples taken 25 g of soil, placed in a centrifuge tube with a capacity of 250 ml and pour 150 ml of water. The mixture was thoroughly stirred with a glass rod for 5 min or with electromechani 1 min

Pop-up larger particles immediately removed. After centrifugation (for this purpose, suitable centrifuge brand SSC-3) for 3 min at 800-1000 rpm water is drained, as in a test tube, add 150 ml of a saturated solution of sodium nitrate. Mix with a stick and again centrifuged 3 minutes

The tubes may be installed on a tripod, topped up with the same solution before the formation of a convex meniscus, served skim glass (6×12 cm) so that it touched the liquid layer. After 15 min the tech removed, on a wet surface add a few drops of 50% aqueous solution of glycerin and mikroskopiruyut. To improve the efficiency of identifying eggs is recommended withdrawal of the drug to repeat 2-3 times. In the foci of tinidazol and echinococcosis detection eggs hampered by the fact that they have a certain stickiness and adhere to soil particles, ill see you in the flotation solution.

The main disadvantage of this method is the low specific gravity of the flotation solution. In addition, a saturated solution of sodium nitrate, as well as alternative solutions proposed in the literature (sodium thiosulfate and sodium nitrate), very quickly crystallize, making it difficult to view samples and reduces the detection of helminth eggs.

The aim of this work was to improve the detection of helminth eggs, ticks and oocysts of protozoa in soil samples, the implementation of a quantitative assessment of the degree of contamination of the soil.

Us using the developed method was carried out comparative analysis of methods of soil: Romanenko and developed a method of identifying helminth eggs, ticks and oocysts of protozoa.

In a sample of sand was artificially made eggs of helminths Ascaris suum, Toxocara canis and Ascaris lumbricoides, protozoa Eimeria tenella, E. maxima, E. acervulina, ticks Knemidocoptes mutans in the amount of 100 eggs or oocysts in g of sand. Eggs were obtained from m the TCI females of worms. The simplest received from the litter spontaneously infected chickens. Knemidocoptes mutans was obtained from scrapings from the affected limbs of patients with birds.

The efficiency of detection of eggs in the soil are statistically significantly different when evaluated using the student test at the significance level of 0.95 (district DU, Mamonov L.M., Astafiev, VA, Zhdanov, S., 2004).

The results of the comparative analysis the following results were obtained.

The developed method allows to identify for 36% of the eggs of Ascaris more, compared with the method Romanenko N.A., due to the use of flotation in multicomponent flotation system, which was the basis of this method. The components offered by us flotation system does not violate the morphology of the eggs of parasites and do not create problems with the morphological identification of the pathogen. Each of them performs a specific function: zinc chloride increases the density of the solution, glycerin reduces the time of crystallization, increasing the quality of research, zinc chloride reduces the cost of analysis. As a rule, the use of such mixtures can improve the detection of eggs, oocysts, and cysts of protozoa, but requires optimization of the composition of the mixture to each specific case (Lutfullin -, Latypov became popular, Kornishina PPM of Helminth-laproscopically research is of animals. Kazan, 2002, 24 C.). Thus, the proposed method allows to significantly increase the efficiency of detection of eggs, cysts and oocysts of protozoa in soil samples compared with the method Romanenko N.A. and can be used in practice and research.

Identification of helminth eggs, ticks, oocysts of protozoa soil samples patented method were carried out according to the following scheme: first, preparing a combined flotation mixture of three ingredients:

1) a saturated solution of zinc chloride (ZnCl 2 kg2on 1 l of water);

2) a saturated solution of sodium chloride (0.42 kg NaCl per 1 l of water).

3) glycerin (H).

Further, these three components are mixed in a ratio of 1:1:1. Pop-up larger particles immediately removed. After centrifugation (for this purpose, suitable centrifuge brand SSC-3) for 3 min at 800-1000 rpm water is drained, and centrifuge cups, add 150 ml three-flotation system. Mix with a stick and again centrifuged 3 minutes

The tubes may be installed on a tripod, topped up with the same solution before the formation of a convex meniscus, served skim glass (6×12 cm) so that it touched the liquid layer. After 20-30 min, the glass is removed and mikroskopiruyut order to improve identification of eggs is recommended withdrawal of the drug to repeat 2-3 times.

Sravnitelnyeharakteristiki detection of eggs of Ascaris suum in soil samples are presented in Table 1.

Table 1
Comparison of efficiency of detection of eggs in soil samples from the artificial introduction of eggs of Ascaris suum method Romanenko N.A. and the patented method
No. soil samplesThe number of eggs of Ascaris suum in the soil sample
RomanenkoPatented method
129
238
3210
4411
5511
627
7312
8211
918
10210
M±M2,6±0,419,7±0,52

The table shows that the patented method was developed to identify 7.1 eggs Ascaris suum more than the method Romanenko.

Comparative efficacy of detection of Toxocara canis eggs in soil samples is presented in Table 2.

Table 2
Comparison of the efficiency determination of eggs in soil samples from the artificial introduction of eggs of Toxocara canis method Romanenko N.A. and the patented method
No. soil samplesThe number of Toxocara canis eggs in the soil sample
RomanenkoPatented method
1113
2314
3410
4412
512
6311
7313
8111
9112
1039
M±M2,8±0,4711,7±0,41

The table shows that the patented method was developed to identify 8 -, 9 Toxocara canis eggs more than the method Romanenko.

Similar results were obtained using human eggs roundworm Ascaris lumbricoides (table 3).

Table 3
Comparison of the efficiency determination of eggs in soil samples from the artificial introduction of eggs of Ascaris lumbricoides method Romanenko N.A. and the patented method
No. soil samplesThe number of eggs of Ascaris lumbricoides in the soil sample
RomanenkoPatentable subject is d
1111
2412
3210
4113
5211
6512
7312
8212
9311
10215
M±M2,5±0,4211,9±0,46

The table shows that the patented method was developed to identify 9.4 eggs Ascaris lumbricoides more than the method Romanenko.

The results of studies of the comparative effectiveness of detection in soil samples oocysts of ameri and eggs Knemidocoptes mites of the genus patent-pending method and method Romanenko N.A. presented in Table the price of 4.

Table 4
Comparison of the effectiveness of the definition of oocysts of ameri and ticks Knemidocoptes mutans in soil samples from the artificial introduction of oocysts of Eimeria tenella, E. maxima, E. acervulina and mites method Romanenko N.A. and the patented method
No. soil samplesRomanenkoPatented method
OocystsEggsOocystsEggs
ameriK. mutansameriK. mutans
1711112
2411511
3331212
411 1313
5221115
6221614
7231111
8411411
9411815
10311610
M±m3,2±0,561,6±0,2813.7±0,8312,4±0,59

The table shows that the patented method was developed to identify 10.5 oocysts of ameri and 10.8 eggs ticks more than the method Romanenko.

Example 1 (lab room No. 8). The sample was collected in the sandbox, p is andlease kindergarten №373 Volga district, Kazan, 11.06.2010 In the yard of the kindergarten can be accessed animals. The total mass of the sample 200,

In the study method Romanenko N.A. helminth eggs was not found.

Then was applied dojinsdirty combined method of soil investigation, patent-pending us.

For the study of the total sample selected 25 g of soil was placed in a centrifuge tube with a capacity of 250 ml and was filled with 150 ml of water. The mixture was thoroughly stirred with a glass rod for 5 minutes

Pop-up larger particles immediately removed. After centrifugation (for 5 min at 1000 rpm), the water is decanted, and the tubes were added 150 ml duringevening flotation mixture. Mixed coli and again centrifuged 5 minutes

Tubes mounted in a tripod, refilled with the same mixture before formation of a convex meniscus, covered skim glass (6×12 cm) so that it touched the liquid layer. After 20-30 min, the glass is removed and mikroskopiruyut. To improve the detection of eggs withdrawal of the drug to repeat 2-3 times.

We have uncovered: eggs sp. Toxocara, Trichocepalus and free-living worm.

Example 2 (lab room No. 9). The sample was collected on the interior of the territory of the house there is free access to the birds. The total mass of the sample 200,

In the study by the method of Romanico N.A. eggs of worms, protozoa and mites, Vaulina was not.

Pat is themim tradingrelated way to study soil were detected oocysts of Eimeria tenella, E. maxima, eggs Thominx collaris, eggs of Ascaridia galli eggs and mites Dermanyssus gallinae and K. mutans.

From the presented data shows that in the newly developed method of identifying eggs can significantly increase the percentage detected in soil eggs soil-transmitted helminths such as human and pig Ascaris, and Toxocara, approximately 4-5 times, compared to the recommended literature method Romanenko N.A. in Addition, in the newly developed method allows to halve the waiting time in the analysis. This makes it justified the use of patent-pending us the method in practice and for research.

Figure 1. Eggs of Ascaris, isolated from the uterus of the worm and detected in the soil sample after artificial make.

Figure 2. Free-living worm, discovered the patented method; magnification ×200.

Figure 3. Egg Trihotel detected in the soil sample patentable method; magnification ×200.

Literature

1. MUK 4. 2. 796 - 99. Methods of sanitary parasitological studies. 2. Lutfullin -, Latypov became popular, Kornishina PPM of Helminth-laproscopically research animals. Kazan, 2002, p.3 24. Savilov DU, Mamonov L.M., Astaf'ev V.A., S. Zhdanov. The application of statistical methods in epidemiological analysis. - M.: "Medpress-inform", 2004. - P.21 - 26.

Detection of helminth eggs, ticks and oocysts of protozoa in soil samples is, namely, that the soil sample weighing 25 g placed in a centrifuge tube with a capacity of 250 ml and pour 150 ml of water, the mixture was thoroughly stirred with a glass rod for 5 min or with electromechani 1 min and centrifuged for 3 min at 800-1000 rpm, the water is drained, and in a test tube, add 150 ml three-flotation system consisting of a saturated solution of zinc chloride (ZnCl 2 kg21 l), sodium chloride (0.42 kg NaCl per 1 l) and glycerol are taken in the ratio 1:1:1, in a test tube topped up with the same solution before the formation of a convex meniscus, the tube serves skim glass so that it touched the liquid layer, after 20-30 min, the glass is removed and mikroskopiruyut, removal of the drug is recommended to repeat 2-3 times.



 

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