Helminthological Research Methods

HELMINTHOLOGICAL RESEARCH METHODS The first specific helminthological research method should be considered the attempt to detect eggs of parasitic worms of the gastrointestinal tract in feces, introduced into practice by Davaine in 1853. This method was improved by Lutz, who in 1893 applied preliminary washing of excrement with water followed by examination of the sediment, which achieved enrichment of the preparation with eggs and clearing of the field of view from foreign elements. In 1906, Bass proposed treating excrement with a saturated solution of calcium chloride, i.e., a liquid of higher specific gravity than the eggs of parasitic worms, which, thanks to this, floated to the surface and were detected by examining the upper layer of the mixture. Subsequently, a large number of methods for processing excrement were proposed, based mainly on these two principles of sedimentation and flotation of eggs. Methods for the quantitative counting of eggs were developed. Investigations began to be carried out not only on excrement but also on other excretions, secretions, and tissues of the human body. Methods for cultivating parasitic worms on special media were proposed. Skryabin developed a method of complete helminthological autopsies, which makes it possible to determine not only the qualitative composition of the helminth fauna of a given individual but also the quantitative one, with an accurate count of all individuals of each species of parasitic worm and an accurate determination of their localization. At the present time, methods of immuno-biological reactions (precipitation, complement fixation reaction, skin reactions, etc.), methods of X-ray diagnostics, ophthalmoscopy, and some others are beginning to find more frequent application for the diagnosis of helminthiases. Method of complete helminthological autopsy of a human. After a preliminary examination of the skin and mucous membranes, followed by the removal of the skin and examination of the subcutaneous tissue, the autopsy is performed. Organ systems are isolated and serous cavities are examined. Groups of muscles (ocular, diaphragm crura, intercostal muscles) are taken for trichinelloscopy, which are examined in the same way as in a biopsy (see below). Synovial cavities, eyes, spinal cord, and brain are opened and examined. Organs of all systems are carefully isolated so as not to cut the parasites, and the examination is begun using the following methods: I. Method of sequential washing and draining of liquid from the sediment until the wash waters are completely transparent. In this way, the following are processed: 1) contents of organs (stomach, all sections of the intestines, gallbladder, eyes; urine; blood from the heart, large vessels, and blood collected during the autopsy in the thoracic and abdominal cavities), 2) parenchymal organs after preliminary crushing with fingers (liver, pancreas, lungs, kidneys, reproductive organs). II. Method of compression examination of scrapings. The mucosa from all cavitary organs (esophagus, stomach, all sections of the intestine, gallbladder, trachea, large bronchi, renal pelvis, ureters, urinary bladder, reproductive organs) is scraped, pressed between two compression glasses, and examined by means of a magnifying glass. III. Method of crushing tissues between glasses (compressure). Layered sections of parenchymal organs (brain and spinal cord; kidneys and reproductive organs can also be used) are crushed between two glasses to the point of transparency. IV. Method of examining the "matrix" (washed contents of the cavities of various organs) alternately on a white and black background in order to collect all parasites visible to the naked eye. V. Method of examining the "matrix" of scrapings and crushed tissues with the help of a magnifying glass. Apparatus and reagents for complete helminthological autopsies. In addition to the usual set of instruments used in pathological-anatomical autopsies, the following are necessary: small scissors with narrow ends for the deepest possible penetration along the passages of the bronchi, bile ducts, etc.; glass cylinders for sequential washing and a stand for them; small baths or photographic trays for isolating organs and viewing the sediment; a dissecting magnifying glass and Petri dishes for viewing the sediment under the magnifying glass; compression glasses (8x24 cm), microscope slides and coverslips. For catching parasites: dissecting needles, eye forceps, brushes, eye pipettes with a rubber bulb. Physiological saline solution when opening the heart, large vessels, and washing blood. Barbagallo's fluid [3% solution of commercial formalin (40% formaldehyde) in physiological saline] for fixing and preserving roundworms. 75% alcohol for fixing and preserving flatworms and thorny-headed worms. Methods for examining excrement are divided into macrohelminthoscopy and microhelminthoscopy of excrement. For macrohelminthoscopy, excrement is mixed with water, or even better with physiological saline, to a liquid consistency and examined in black trays under bright lighting. When searching for small forms, the method of sequential washing of excrement until the wash waters are transparent is used, and the sediment is examined both in trays and under a magnifying glass. For microhelminthoscopy, there are many methods, the most common of which are given below. Microhelminthoscopy should always be carried out in parallel with macrohelminthoscopy of excrement, and macrohelminthoscopy is sometimes used in isolation, for example, during control examination of excrement after dehelminthization. Since the majority of microhelminthoscopic methods have the task of detecting eggs of parasitic worms in various excretions and secretions, they are called methods of helmintho-ovoscopy. Native smear method. A particle of excrement, smaller than a millet grain, is ground on a microscope slide using a matchstick with a drop of 50% glycerin solution (or physiological saline) and examined under a coverslip. It is mainly an auxiliary method. Telemann's method in the modification of Miyagawa. Excrement, the size of a hazelnut, is thoroughly "shaken" in a test tube or, better, ground in a mortar with equal amounts of 50% hydrochloric acid and sulfuric ether; the mixture is filtered through a sieve, centrifuged, and the sediment is examined. Kofoid and Barber's method in the modification of Fülleborn (incorrectly called the Fülleborn method). Excrement is thoroughly mixed in a beaker or other vessel with a saturated solution of table salt (400 g of salt is required to saturate 1 liter of water) in a ratio of 1:20 (approximate). Coarse parts of excrement that did not yield to grinding and floated to the surface of the mixture immediately after mixing are removed with a spatula. The mixture should stand for 1-2 hours (and not 1/4-3/4 of an hour, as Fülleborn recommends), after which the surface layer containing the floated eggs is removed with a platinum or simple wire loop, about 0.8 mm in diameter, bent at a right angle to the handle, by means of contact of the loop with the surface layer of the mixture. The film remaining in the loop is transferred to a microscope slide and examined. Darling's method is a combined one, based on the principles of both sedimentation and flotation. Excrement is mixed with water to a semi-liquid consistency, the mixture is centrifuged, the liquid is drained, and a saturated solution of table salt and glycerin in equal amounts is added to the sediment, after which the mixture is centrifuged again. Eggs floating on the surface are removed with small pieces of cotton wool, which are examined within the first hour (before the eggs have had time to clear). It should be noted that the eggs of trematodes and tapeworms float with more difficulty than those of nematodes and cestodes, so flotation methods should be supplemented with at least the smear method. Stoll's method for quantitative counting of eggs. 3 g of excrement are weighed into a test tube marked 45 cubic cm; it is filled to the 45 cubic cm mark with a decinormal solution of sodium hydroxide; it is thoroughly shaken with glass beads until a uniform emulsion is obtained; 0.15 cubic cm is quickly drawn from the test tube with a graduated pipette and the mixture is poured onto a microscope slide, covered with a 22x44 mm coverslip, and the eggs are counted. The number of eggs present in the preparation, multiplied by 100, is equal to the number of eggs contained in 1 g of excrement. For greater accuracy, Stoll recommends performing a double examination. Among other methods of quantitative egg counting, one can mention the Hung method, based on the flotation principle, and the Lane method, a combination of flotation and sedimentation principles. In view of the fact that the examination of excrement yields completely unreliable results regarding the detection of Enterobius vermicularis (pinworm) eggs, there are special, more accurate methods of examination for enterobiasis. Of these, the most common is the scraping method from the perianal folds. A scraping is taken from the mucosa surrounding the anus and from the area of skin in contact with it using a diagonally cut matchstick moistened in a 50% glycerin solution. The scraping is transferred to a drop of 50% glycerin solution on a microscope slide, where it is thoroughly scraped with another matchstick or the edge of a microscope slide.

It is advisable to use the scraping method for examination for enterobiasis, as well as for the subungual spaces, perineum, vulva (both its mucous membrane and secretions), etc. Sputum examination. Sputum is placed on a glass slide, covered with another (compression method), and examined macroscopically both against a black and white background and by means of a magnifying glass. For detailed examination, the sputum preparation on the glass slide is viewed with the help of a microscope. Sputum can be examined by the smear method and by the Telemann method. Examination of duodenal juice and gallbladder bile. Mucus flakes are selected and examined under a microscope; duodenal juice or gallbladder bile is mixed with a large amount of ether and centrifuged; the sediment is examined. Urine examination: the sediment is examined after centrifugation for the presence of parasitic worms or their elements (eggs, larvae, fragments, etc.). The chemical reaction proposed by Efimov for the detection of helminthic toxins in urine by treating it with mercurous nitrate proved to be non-specific. Under special indications, nasal mucus, gastric juice, and vomitus, transudates and exudates, and all kinds of punctates are subjected to examination. Depending on the need, the following are used: smear, simple centrifugation, the Telemann method. Blood examination is performed either for the direct detection of parasitic worms (microfilariae in filariasis) or for studying changes in blood composition in various helminthic diseases. Microfilariae can be studied in a living state in a drop of blood under a coverslip, the edges of which are greased with petroleum jelly to protect the preparation from drying out. Blood mixed with sodium citrate to prevent clotting can be pre-centrifuged, and then the microfilariae are concentrated in the sediment. It is also possible to use vital staining of microfilariae, for which a little dye prepared in physiological saline (best of all Azure II with a weak solution of eosin or neutral red) is added to a fresh drop on a microscope slide. To prepare permanent preparations of microfilariae, thick blood smears are made, which are fixed in the usual way, or coverslips with fresh, still moist blood smears are placed in 60-70% heated alcohol. For staining, Böhmer's hematoxylin, hydrochloric acid carmine, methylene blue with fuchsin, Giemsa solution, etc., are used. The most successful staining is provided by the Romanowsky-Giemsa method and Azure II. For special examination for trichinosis and cysticercosis, tissue biopsy (muscles, intermuscular connective tissue, subcutaneous tissue) is resorted to. Small pieces of muscle are teased apart with dissecting needles into individual fibers, compressed strongly between two microscope slides (even better in special compressors), and examined with the help of low-power magnification of a microscope. If cysticercosis is suspected, the subcutaneous tissue and connective tissue layers of the muscles are carefully examined for a more detailed study of suspicious formations. Helmintho-ovoscopy, as well as the examination of larvae of parasitic worms and their fragments, are performed with the help of a microscope [eyepiece 3 or 4, objective 3 (or AA) with a somewhat darkened field of view]. Suspicious formations and eggs for accurate diagnosis are examined with the same eyepiece, with objective 447

HELMINTHOLOGY

448 6, 7, or 8 (or DD). The material under investigation must be examined under a coverslip. For accurate diagnosis, in many cases, it is necessary to resort to measuring the length and width of eggs, i.e., to so-called helmintho-ovometry (see Micrometry). Beginners are recommended to use helmintho-ovometry even when examining common forms. Measurement of eggs should be performed using objectives 6-8 or their equivalent in terms of magnification. The maximum length and width of the most mature eggs are measured, i.e., those located closer to the vulvar opening (when measuring eggs contained in the reproductive organs of the parasite). In cestodes, in addition to the egg, the diameters of the oncosphere and the length of the hooks are measured. The method of cultivating parasitic worms on special media was first proposed by Loos. Excrement, diluted with water to a semi-liquid consistency, is mixed in a Petri dish with animal charcoal ground into a powder, taken in double quantity. The culture is placed for 4-5 days at 30°. The larvae that have hatched from the eggs and grown are washed off with water; the water is filtered; the filter, on the walls of which the larvae collect, is folded into the shape of a pouch and lowered into a cylinder with water, into which the larvae penetrate, forming a 'pure culture.' Subsequently, many authors modified this method, suggesting taking calcined earth or agar instead of animal charcoal, on which pieces of excrement were placed (Fülleborn). In the latter case, the presence of larvae was detected macroscopically by the zigzag paths left by them during movement. Methods of processing helminthological material for study. To study the details of the structure of parasitic worms after their fixation, the following methods are used. Nematoda are cleared in lactic acid. Cestoda are stained in a weak (5-10%) solution of Grenacher-Delafield hematoxylin or Mayer's alum hematein. Trematoda are stained with alum carmine. After staining, the parasites are mounted in Canada balsam according to the rules of histological technique. To preserve excrement containing eggs, it is diluted with water to a semi-liquid consistency and mixed with 10% formalin or with a mixture of equal amounts of 5% formalin and 5% glycerin. To prepare microscopic preparations of eggs, they are mounted on a microscope slide in a drop of warmed glycerin-gelatin (gelatin - 2 g; Aq. destill. - 12 cc; glycerin - 7 g; a few drops of Acid. carbol.; mix well in a water bath) and covered with a coverslip. Less stable eggs (for example, Hymenolepis nana, Trichostrongylidae, Ankylostoma, Necator) are better preserved in Barbagallo's fluid after preliminary processing of the excrement by the Telemann method; the coverslip is edged with thick Canada balsam, which is heated for thinning.