Trichinosis
The presumptive diagnosis of trichinosis is often based on a combination of facts: history of ingestion of raw or rare pork, walrus meat, horse meat, or bear meat; diarrhea followed by edema and muscle pain; and the presence of eosinophilia. Usually, by the time the patient is symptomatic, the suspected food is no longer available for examination. The diagnosis may be confirmed by finding larval Trichinella spp. in a muscle biopsy specimen. The encapsulated larvae (which may be few) are easily seen in fresh muscle if small pieces are pressed between two slides and examined under the microscope (Fig. 6.19). Larvae are usually most abundant in the diaphragm, masseter muscle, or tongue and may be recovered from these muscles at necropsy. Routine histologic sections can also be prepared.
Figure 6.19 Trichinella spp. larvae in a squash preparation of muscle biopsy tissue; note the encysted larvae. doi:10.1128/9781555819002.ch6.f19
A portion of the specimen can be digested in artificial digestive fluid at 37°C, with the larvae recovered by centrifugation; however, very young larvae might also be digested.
Artificial Digestive Fluid
1. Prepare the tissue by grinding (with a tissue grinder or commercial meat grinder or blender). The tissue should be medium grind; do not overgrind, particularly in a blender.
2. Add the tissue to the digestive fluid in the ratio of 1 part tissue to 20 parts fluid in an Erlenmeyer flask.
3. Place the flask in an incubator at 37°C, on a shaker table or magnetic stirrer if available; leave for 12 to 24 h.
4. Add warm water (37°C) to the digestive fluid mixture. (Add enough water to triple the original volume.)
5. Pour the diluted mixture into a Baermann funnel, and add water up to the screen.
6. Allow the mixture to stand for 1 to 2 h; the larvae will settle out in the lower part of the funnel.
7. Remove a few drops of fluid, and examine the material under the microscope (10× objective) for the presence of larvae.
8. If no larvae are present, centrifuge 50 ml and examine the sediment.
Cestode Larval Stages
Human infection with any of the larval cestodes may present diagnostic problems, and frequently the larvae are referred for identification after surgical removal. In addition to Echinococcus granulosus (hydatid disease) and the larval stage of Taenia solium (cysticercosis), other larval cestodes occasionally cause human disease. The larval stage of tapeworms of the genus Multiceps, a parasite of dogs and wild canids, is called a coenurus and may cause human coenurosis. The coenurus resembles a cysticercus but is larger and has multiple scolices developing from the germinal membrane surrounding the fluid-filled bladder. These larvae occur in extraintestinal locations, including the eye, central nervous system, and muscle (see chapter 25).
Human sparganosis is caused by the larval stages of tapeworms of the genus Spirometra, which are parasites of various canine and feline hosts; these tapeworms are closely related to the genus Diphyllobothrium. Sparganum larvae are elongated, ribbonlike larvae without a bladder and with a slightly expanded anterior end lacking suckers. They are usually found in superficial tissues or nodules, although they may cause ocular sparganosis, a more serious disease (see chapter 25).
The diagnosis of larval cestodes is frequently facilitated by the recognition of prominent calcareous corpuscles occurring in the tapeworm tissue; specific identification usually depends on referral to specialists.
Schistosomiasis
Often when a patient has an old, chronic infection or a light infection with Schistosoma mansoni or S. japonicum, the eggs may not be found in the stool and an examination of the rectal mucosa may reveal the presence of eggs. The fresh tissue should be compressed between two microscope slides and examined under the low power of the microscope (low-intensity light). Critical examination of these eggs should be made to determine whether living miracidia are still found within the egg. Treatment may depend on the viability of the eggs; for this reason, the condition of the eggs should be reported to the physician.
Mucosa from the bladder wall may reveal eggs of Schistosoma haematobium when they are not being recovered in the urine. As with rectal biopsy specimens, the eggs in the bladder wall should be checked for viability (fresh, unfixed tissue) (Fig. 6.20).
Figure 6.20 Schistosoma haematobium eggs seen in a squash preparation of a bladder biopsy specimen. Note the pointed terminal spine. (Armed Forces Institute of Pathology photograph.) doi:10.1128/9781555819002.ch6.f20
Viability Testing of Schistosome Eggs
1. With careful observation of the egg, using the 40× objective (low-intensity light), the cilia of the flame cells of the miracidium within the shell can be seen to move in a rapid flickering motion.
2. The eggs within the tissue can be removed (carefully tease the tissue apart in saline solution) and subjected to a hatching procedure. If the miracidia are released from the egg and swim to the top of the hatching flask, this movement is also proof of viability. (The procedure can be found in chapter 4.)
Digestion Procedure in Diagnosis of Schistosomiasis
Small pieces of tissue may be digested in 4% potassium hydroxide (4% sodium hydroxide may be used) for 2 to 3 h at 60 to 80°C. The material may then be concentrated by sedimentation or centrifugation and examined under the microscope for eggs (low power with low-intensity light).
References
1. Schwartz DA, Bryan RT, Hewanlowe KO, Visvesvara GS, Weber R, Cali A, Angritt P. 1992. Disseminated microsporidiosis (Encephalitozoon hellem) and acquired immunodeficiency syndrome—autopsy evidence for respiratory acquisition. Arch Pathol Lab Med 116:660–668. PMID 1616428
2. Current WL, Garcia LS. 1991. Cryptosporidiosis. Clin Microbiol Rev 4:325–358. PMID 1889046
3. Garcia LS. 1999. Practical Guide to Diagnostic Parasitology. ASM Press, Washington, DC.
4. Garcia LS, Shimizu RY. 1997. Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens. J Clin Microbiol 35:1526–1529. PMID 9163474
5. Siddons CA, Chapman PA, Rush BA. 1992. Evaluation of an enzyme immunoassay kit for detecting Cryptosporidium in faeces and environmental samples. J Clin Pathol 45:479–482. PMID
