WS/T 570-2017Detection of intestinal helminths - The Kato-Katz method (English PDF)

4.1 Reagents

Composition and preparation of clear liquid. 100ml of pure glycerin, 1ml of 3% malachite green (or methylene blue) aqueous solution and 100ml of distilled water Prepared.

4.2 Materials

4.2.1 Plastic quantitative plate

The specification is 30mm×40mm×1mm, the central hole is truncated cone shape, the upper bottom radius is 3mm, the lower bottom radius is 4mm, the height is 1mm, and the volume is 38.75mm 3.

4.2.2 Scraper

60mm × 6mm × 2mm, one end is flat, the other end is a beveled surface, the beveled surface is 8.23mm long.

4.2.3 Nylon silk

80 mesh, that is, the number of sieve holes per inch (25.4mm) length is 80, cut into 8cm×8cm size.

4.2.4 Hydrophilic transparent cellophane

40µm thick, cut into 25mm×40mm size, soak in clear liquid for more than 24h before use.

4.2.5 Slides

76.2mm × 25.4mm × 1mm.

5.1 Sample collection

The test sample is fresh stool. Collect about 30g of the subject's feces and store them in a wide-mouthed, leak-proof, closed container.

Mark the subject's basic information (name, number, date of delivery) on the outside of the container. The collected samples should be sent for inspection within 24h, If it is not detected in time, it should be stored at 4°C.

5.2 Improved Kato Thick Sheet Production

5.2.1 Take a clean glass slide and mark one end with the sample number. Place the plastic quantitative plate on the middle of the glass slide with the small hole facing up.

5.2.2 Lay the nylon silk flat on the feces and spread it out. Use a scraper to lightly press the nylon silk to make the nylon silk and the feces closely fit, and then use the scraper Scrape the feces over the nylon silk.

5.2.3 Fill the fecal sample scraped by nylon silk into the central hole of the quantitative plate until it is filled and smoothed.

5.2.4 Remove the quantitative plate vertically upwards to leave the fecal sample on the slide.

5.2.5 Take a piece of hydrophilic transparent cellophane that has been soaked, shake off the excess soaking liquid, and cover it on the feces. Take another piece the clean glass slides are crossed vertically and lightly to press the fecal sample evenly, so that the feces under the hydrophilic transparent cellophane are evenly spread without overflowing the glass slide A round fecal membrane of uniform thickness is formed, with a diameter of about 2 cm.

5.2.6 Fix the hydrophilic transparent cellophane with the thumb, and gently move the slide used to press the fecal sample to remove the modified Kato the thick smear is placed at room temperature to make it transparent, and the transparent time is not easy to exceed 2h.

5.3 Microscopic examination

Place the transparent modified Kato thick smear under a biological microscope for microscopic examination, and follow a certain rule under a 10× objective lens, such as from top to bottom, Check the whole film from left to right, and observe under a 40× objective lens for further identification.

5.4 Results judgment

5.4.1 According to the comprehensive characteristics of the egg size, shape, color, egg shell, content, egg cover, and small spines in the modified Kato thick smear Co-determined insect species (see Appendix A).

5.4.2 Count the number of eggs in the modified Kato thick smear, calculate the EPG, and determine the intensity of the infection (see Appendix B).

5.5 Notes

5.5.1 After the fecal membrane is transparent, microscopic examination should be timely. If the transparency is too high, the thin-shelled eggs will be deformed and difficult to identify, which will easily lead to missed inspections or misjudgments.

Generally, the modified Kato thick sheet should not be placed for more than 2 hours at room temperature of 25°C and 75% humidity. If the air humidity is high and the temperature is low, the storage time can be extended appropriately. The air is dry, the temperature is high, and the storage time is appropriately shortened. When transparent, do not try to shorten the transparent time Put the modified Kato thick slices in the oven or expose them to the sun.

5.5.2 In special studies on prevention and treatment such as drug efficacy assessment, the calculation of EPG must be multiplied by the stool coefficient (formed stool 1, semi-formed stool Stool 1.5, soft stool 2, porridge-like stool 3, watery stool 4), get the number of eggs per gram of feces. Since the total amount of stool in children is less than that in adults, this child contains more eggs per unit volume of feces than adults, so adults should be used as the standard and proportionally reduced, that is, children's feces the number of eggs is multiplied by 25% for 1 to 2 years old, 50% for 3 to 4 years old, 75% for 5 to 10 years old, and no reduction for those over 11 years old.

Appendix A

(Informative appendix) Identification of worm eggs in human feces A.1 Egg types the parasite eggs that can be detected by the improved Kato thick smear method are. vermilike roundworm eggs, trichocarpus eggs, vermicelli Intestinal nematode eggs, hookworm eggs (Ancylostoma duodenum eggs, Ancylostoma americana eggs and Ancylostoma eggs), Taenia mansoni Eggs, Schizocephalus eggs, Taenia eggs (Taenia solium eggs, Taenia saginata eggs, Taenia asiatica eggs), Hymenocystis miniature eggs, Shrink Hymenoptera eggs, Siberian tapeworm eggs, Clonorchis sinensis eggs, Fasciola brucei eggs, Paragonimus wessonii eggs, Japanese blood Fluke eggs, Schistosoma mansoni eggs, Fasciola eggs (fasciola hepatica eggs, Fasciola macrofasciatus eggs), Fasciola hepatica eggs, Echinochosoma Fluke eggs, etc. The above-mentioned eggs can be identified under a microscope by their shape, size, color, content, egg cover structure, etc. (see

Table A.1).

B.1 Full slice count Every modified Kato thick smear found ascaris lumbricoides eggs, whipworm eggs, hookworm eggs, Clonorchis sinensis eggs, Japan all worm eggs such as schistosome eggs must be counted. The eyepieces of the microscope used in the microscopy are unified as a 10× lens.

B.2 Estimate the number of eggs B.2.1 Calculation steps when the number of eggs of a certain kind in a modified Kato thick smear reaches thousands or even tens of thousands, it takes time to count.

The number of eggs in thick smears shall be as follows. each smear shall firstly check several fields at random, if the number of eggs of a certain kind in each field is 10 Above, you can temporarily stop counting the eggs. After reading the number of other eggs in the entire film, use the fixed-field spot check method to calculate the number of eggs in the entire film.

Specific steps are as follows.

B.2.1.1 According to Figure B.1, the distribution of fixed spot check 10 visual fields, and calculate the total number of eggs in the 10 visual fields

Figure B.1 Schematic diagram of the distribution of 10 fields of view

B.2.1.2 Calculate the field of view of the smear film, and then calculate the number of eggs in the whole film According to certain rules, such as from left to right, top to bottom, then right to left, line by line, one field of view after another Use the propeller to move the smear to count the number of fields of view of the entire fecal membrane, then. the total number of eggs in the entire fecal membrane = the average number of eggs in the 10 visual fields × the entire feces the total number of membrane fields.

B.2.2 Infectivity classification the average weight of feces contained in each hole of the plastic quantitative plate is 41.67mg, and the number of eggs obtained from each modified Kato thick smear is multiplied by 24.

The number of eggs per gram of feces.

Number of eggs per gram of feces (EPG) = average number of eggs obtained from each modified Kato thick smear × 24 See Table B.1 for the grading standard of infection degree of important human parasites.