Diagnostic Medical Parasitology. Lynne Shore Garcia
based on a number of variables mentioned above. It is unrealistic to assume or state that one approach is applicable for every laboratory; however, it is important to thoroughly understand the options within your test menu and to convey this information to your clients once your approach has been selected for implementation. Prior discussion with clients, written educational memos, meetings, and examples of revised report formats are highly recommended prior to implementation.
A series of three specimens as indicated above should be submitted on separate days; if possible, the specimens should be submitted every other day; otherwise, the series of three specimens should be submitted within no more than 10 days. If a series of six specimens is requested, the specimens should also be collected on separate days or within no more than 14 days. Many organisms, particularly the intestinal protozoa, do not appear in the stool in consistent numbers on a daily basis, and the series of three specimens is considered a minimum for an adequate examination. It is inappropriate for multiple specimens to be submitted from the same patient on the same day. One possible exception would be stool collections from a patient who has severe, watery diarrhea such that any organisms present might be missed because of the tremendous dilution factor related to fluid loss. Even under these circumstances, acceptance of more than one specimen per patient per day should not be routine but should be done only after consultation with the physician. It is also not recommended for the three specimens to be submitted one each day for three consecutive days; however, use of this collection time frame would not be sufficient cause to reject the specimens. Adequate spacing between specimens helps to provide parasite recovery within the recommended time frames.
Although the recommended number of stool specimens is three, laboratories have been more willing to accept two specimens, primarily because of cost savings and the assumption that if the patient is symptomatic, confirmation of any organisms present is just as likely to be possible from two specimens as from three specimens. However, it is important that clients understand the pros and cons of two compared with three stools. Both collection approaches are being used by diagnostic laboratories.
Specimen Type, Specimen Stability, and Need for Preservation
Fresh specimens are mandatory for the recovery of motile trophozoites (amebae, flagellates, or ciliates). The protozoan trophozoite stage is normally found in cases of diarrhea; the gastrointestinal tract contents are moving through the system too rapidly for cyst formation to occur. Once the stool specimen is passed from the body, trophozoites do not encyst but may disintegrate if not examined or preserved within a short time after passage. The time limit recommendations listed below are most relevant for the intestinal protozoa; most helminth eggs and larvae, coccidian oocysts, and microsporidian spores survive for extended periods. However, no one can predict which organisms will be present in the stool specimens; therefore, it is important to use the most conservative time frames for parasite recovery.
Liquid specimens should be examined within 30 min of passage, not 30 min from the time they reach the laboratory. If this general time recommendation of 30 min is not possible, the specimen should be placed in one of the available fixatives. Soft (semiformed) specimens may contain a mixture of protozoan trophozoites and cysts and should be examined within 1 h of passage; again, if this time frame is not possible, preservatives should be used. Immediate examination of formed specimens is not as critical; in fact, if the specimen is examined at any time within 24 h after passage, the protozoan cysts should still be intact (5, 16).
In review, remember that trophozoites only are usually found in liquid specimens, both protozoan trophozoites and cysts can be recovered in soft specimens, and generally cysts only are recovered in formed specimens. The time limits mentioned above are merely guidelines; however, if fresh specimens remain unpreserved for longer times before examination, many if not all organisms may disintegrate or become distorted. Fecal specimens should never be incubated or frozen prior to examination using routine microscopy. When the acceptance criteria for specimen collection are not met, the laboratory should reject the specimen and request additional specimens.
Because there is often a time lag from the time of specimen passage until receipt in the laboratory, many clinicians, clinics, and inpatient wards use a specimen collection system that includes stool preservatives (Table 2.3). A number of commercial systems are available with many preservative choices; the use of such systems has become routine for many institutions, and some request a custom collection kit that may contain several types of preservatives for stool specimens, depending on the tests normally ordered by the clinicians that they service. Specific information concerning collection kit components is provided in Appendix 1.
KEY POINTS—STOOL SPECIMEN COLLECTION
1. Occupational Safety and Health Act regulations (including standard precautions) should be used for handling all specimens.
2. Interfering substances (oil-based laxatives, barium, antibiotics) should be avoided when stool specimens are collected.
3. Contamination with urine or water should be avoided.
4. Recommendation for collection: three specimens collected, one every other day or within a 10-day time frame.
Note: There are some exceptions; see Table 2.1.
5. Liquid stool: examine or preserve within 30 min of passage (trophozoites). Soft stool: examine or preserve within 1 h of passage (trophozoites and cysts). Formed stool: examine or preserve within 24 h of passage. Note that Dientamoeba fragilis trophozoites can be found in formed stool specimens as well as liquid specimens.
There are a number of reasons why a lag time may occur from the time of specimen passage until examination in the laboratory (e.g., the workload in the laboratory or the transit distance or time for the specimen to reach the facility). To preserve protozoan morphology and to prevent the continued development of some helminth eggs and larvae, the stool specimens can be placed in preservative immediately after passage (by the patient using a collection kit) or once the specimen is received by the laboratory. There are several fixatives available; the more common ones, including formalin, Merthiolate (thimerosal)-iodine-formalin (MIF), sodium acetate-acetic acid-formalin (SAF), Schaudinn’s fluid (with or without polyvinyl alcohol [PVA]), the single-vial systems, and the Universal Fixative, are discussed in detail (Table 2.4). Regardless of the fixative selected, adequate mixing of the specimen and preservative is mandatory. A flow diagram for preservation and processing is shown in Fig. 2.3.
Figure 2.3 Flow diagram for preservation and processing of stool specimens. As mentioned in the text, the examination of fecal specimens using the ova and parasite examination is not considered complete unless a concentration and a permanent stained smear are examined for every specimen submitted to the laboratory. For a fresh specimen, a direct wet mount should be performed if the specimen is very soft to liquid; the complete ova and parasite examination would include the direct wet mount, the concentration, and the permanent stained smear. If the specimen is submitted in preservative, the direct wet mount should be eliminated (no motility is possible); the complete ova and