Diagnostic Medical Parasitology. Lynne Shore Garcia
Modified Kinyoun’s acid-fast stain (cold method) Modified Ziehl-Neelsen acid-fast stain (hot method) Carbol fuchsin negative stain for Cryptosporidium (from W. L. Current) Rapid safranin method for Cryptosporidium Rapid safranin method for Cyclospora, using a microwave oven Auramine O stain for coccidia (from Thomas Hänscheid) Modified trichrome stain for the microsporidia (Weber—green) Modified trichrome stain for the microsporidia (Ryan—blue) Modified trichrome stain for the microsporidia (Kokoskin—hot method) Acid-fast trichrome stain for Cryptosporidium and the microsporidia
If the consistency of a stool specimen can be determined (formed, soft, or liquid), this information may give an indication of the organism stages that might be present. Trophozoites (potentially motile forms) of the intestinal protozoa are usually found in liquid specimens; both trophozoites and cysts might be found in a soft specimen; and the cyst forms are usually found in formed specimens. However, there are always exceptions to these general statements. Coccidian oocysts and microsporidian spores can be found in any type of fecal specimen; in the case of Cryptosporidium spp., the more liquid the stool, the more oocysts that are found in the specimen. Helminth eggs may be found in any type of specimen, although the chances of finding eggs in a liquid stool are reduced by the dilution factor. Tapeworm proglottids may be found on or beneath the stool on the bottom of the collection container. Adult pinworms and Ascaris lumbricoides are occasionally found on the surface or in the stool.
The presence of blood in or on the specimen may indicate several things and should always be reported. Dark stools may indicate bleeding high in the gastrointestinal tract, and fresh (bright red) blood most often is the result of bleeding at a lower level. In certain parasitic infections, blood and mucus may be present. Soft or liquid stool accompanied by blood is more suggestive of an amebic infection; these areas of blood and mucus should be carefully examined for the presence of trophic amebae. Occult blood in the stool may or may not be related to a parasitic infection and could result from a number of different conditions. Ingestion of various compounds may give a distinctive color to the stool (iron, black; barium, light tan to white).
Many laboratories prefer that stool specimens be submitted in some type of preservative. Rapid fixation of the specimen immediately after passage (by the patient) provides an advantage in terms of recovery and identification of intestinal protozoa. This advantage (preservation of organisms before distortion or disintegration) is thought to outweigh the limited motility information that might be gained by examining fresh specimens as direct wet mounts. Other laboratories still request a collection system that includes both a preserved specimen and the remainder of the fresh stool. Certainly cost is a factor, because several vials in the collection system cost more than a single vial containing preservative. Each laboratory will have to decide for itself, often basing the decision on the types of procedures ordered by the physicians who use the laboratory service, the test method selected (traditional methods, immunoassay detection kits, or molecular methods), and the lag time between specimen collection and submission to the laboratory.
With increased emphasis on continuous quality improvement, managed-care contracts, cost containment, and the clinical relevance of diagnostic test results generated, compliance with specimen acceptance or rejection criteria has become more important and a necessary part of overall quality performance. The generation of patient data begins with the quality of the specimen; anything that is done to compromise that quality should not be acceptable within the laboratory setting.
If the specimen has not been preserved immediately after passage, it is important to know the age of the specimen when it reaches the laboratory. Freshly passed specimens are necessary for the detection of trophic amebae, flagellates, and ciliates. Liquid specimens must be examined within 30 min of passage (not 30 min from the time the specimen reaches the laboratory or is clocked in by the computer). Soft specimens should be examined within 1 h of passage. Immediate examination of a formed specimen is not as critical; however, if the stool cannot be examined on the day of collection, portions of the specimen should be preserved. In a routine laboratory setting, these time frames are often neither practical nor possible. Thus, the routine use of stool preservatives for diagnostic parasitology is highly recommended and has been widely accepted.
Microscopic Examination (Ova and Parasite Examination)
The microscopic examination of the stool specimen, normally called the ova and parasite examination, consists of three separate techniques: the direct wet smear, the concentration, and the permanent stained smear. Each of these methods is designed for a particular purpose and forms an integral part of the total examination (1–22, 49). With increased emphasis on proper specimen collection and cost containment, the approach to the ova and parasite examination has changed somewhat during the last few years. Many laboratories are requesting that all fecal specimens be collected in preservatives prior to delivery to the laboratory to decrease the lag time between specimen passage and fixation, thus providing better organism morphology and subsequent identification.
Because preserved organisms do not exhibit motility, the direct wet smear is no longer considered a mandatory part of the routine ova and parasite examination. However, if fresh fecal specimens are delivered to the laboratory, the direct wet smear should be performed, particularly on liquid or very soft stools.
In addition to normal specimen debris, the microscopic examination of fecal material may reveal the following:
1. Trophozoites and cysts of intestinal protozoa
2. Oocysts of coccidia and spores of microsporidia
3. Helminth eggs and larvae
4. Red blood cells (RBCs), which may indicate ulceration or other hemorrhagic problems
5. White blood cells (WBCs), specifically polymorphonuclear leukocytes (PMNs), which may indicate inflammation
6. Eosinophils, which usually indicate the presence of an immune response (which may or may not be related to a parasitic infection)
7. Macrophages, which may be present in bacterial or parasitic infections
8. Charcot-Leyden crystals, which may be found when disintegrating eosinophils are present (and may or may not be related to a parasitic infection)
9. Fungi (Candida spp.) and other yeasts and yeastlike fungi
10. Plant cells, pollen grains, or fungal spores, which may mimic some helminth eggs, protozoan cysts, coccidian oocysts, or microsporidial spores
11. Plant fibers or root or animal hairs, which may mimic helminth larvae
Normal mixing in the intestinal