D. E. histolytica trophozoites measure 10 to 60 µm and demonstrate directional motility by extruding hyaline, finger-like pseudopodia from the cytoplasm. Cysts are not usually found in cultures.
E. Trophozoites are uninucleate and characterized by finely granular, uniform, evenly distributed peripheral chromatin. The nucleolus is small and usually centrally located but may be eccentric.
3. Depending on its use, the microscope(s) may need to be calibrated (within the last 12 months), and the original optics used for the calibration should be in place on the microscope(s). The calibration factors for all objectives should be posted on the microscope for easy access.
Note If the tubes containing fecal material are positive for amebae after 48 h of incubation, confirm the identification with the permanent stained smear. If the tubes do not show any amebae, subculture the contents of the tubes as described above and incubate for an additional 48 h. If the tubes are still negative for amebae, report the specimen as negative and discard the tubes. Even when the culture system is within quality control guidelines, a negative culture is still not definitive in ruling out the presence of E. histolytica. Xenic cultures of E. histolytica serve only as a supplemental procedure and never replace the primary diagnosis by microscopic examination of concentration sediments and permanent stained smears. Axenic culture is used to maintain quality control strains and for research purposes.
Specimens would include cerebrospinal fluid (CSF), biopsy tissue, and autopsy tissue of the brain; for Acanthamoeba spp., corneal scrapings or biopsy material, contact lenses and contact lens paraphernalia such as lens cases and solutions, skin abscess material, ear discharge, or feces can also be used. All clinical specimens should be processed within 24 h; 2 to 3 h is recommended. However, eye-related specimens can be shipped by mail with apparently few problems (18–21). The procedure for growing Naegleria and Acanthamoeba from clinical specimens involves the use of a nonnutrient agar spread with E. coli or some other nonmucoid bacteria. Amebas begin feeding on bacteria and soon grow to cover the agar surface in 1 to 2 days at 37°C. The presence of the protozoa can be confirmed by examining the agar surface using an inverted microscope or with a conventional microscope by inverting the plate on the stage and focusing through the agar with a 10× objective (Fig. 8.1).
Diagnostic methods include direct microscopy of wet mounts of CSF or stained smears of CSF sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histologic assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material. Immunocytochemistry, chemifluorescent-dye staining, PCR, and analysis of DNA sequence variation also have been used for laboratory diagnosis. Several approaches to specimen handling and diagnostic methods can be seen in Table 8.1.
Environmental Issues
There continue to be ongoing discussions and publications regarding the association of free-living amebae and their intracellular bacterial flora, particularly within the context of environmental transmission of infection, contamination of equipment, and overall environmental concerns. The recognition that Acanthamoeba spp. can sequester a variety of bacteria with known potential for causing human disease suggests that these amebae serve as reservoirs for bacterial pathogens. The increase in the reported incidence of Acanthamoeba infections may be due to greater recognition of the disease potential of these amebae. Also, a number of factors may account for an increased incidence of infection, such as a large number of HIV-infected individuals and more patients undergoing chemotherapy or immunosuppressive therapy for organ transplantation (22).
Acanthamoeba Medium
For the isolation of Naegleria or Acanthamoeba spp. from tissues or soil samples, the following procedure is recommended.
Acanthamoeba Medium
Page’s saline (10×)
1. Autoclave at 121°C for 15 min.
2. Store refrigerated in a glass bottle for up to 6 months.
Nonnutrient agar
1. Dissolve agar in Page’s saline and distilled water with gentle heating; stir or swirl.
2. Aliquot 20 ml into screw-cap tubes (20 by 150 mm).
3. Autoclave at 15 lb/in.2 for 15 min; label deeps with 12-month expiration date, and store in the refrigerator.
4. Melt agar deeps, and pour into petri dishes as needed. Plates may be stored in the refrigerator for up to 3 months.
Monoxenic culture
1. Remove the nonnutrient agar plates from the refrigerator, and place them in a 37°C incubator for 30 min.
2. Add 0.5 ml of ameba saline to a slant bacterial culture of E. coli or Enterobacter aerogenes. Gently scrape the surface of the slant (do not break the agar surface). Suspend the bacteria uniformly by gently pipetting with a Pasteur pipette, and add 2 or 3 drops of this suspension to the middle of the warmed agar plate. Spread the bacteria on the surface of the agar with a bacteriological loop.
3. Inoculate the specimen on the center of the agar plate as described below.
A. For CSF samples, centrifuge the CSF at 250 × g for 10 min. With a sterile serologic pipette, carefully transfer all but 0.5 ml of the supernatant to a sterile tube and store at 4°C (for possible future use). Mix the sediment in the rest of the fluid, and, with a Pasteur pipette, place 2 or 3 drops in the center of the nonnutrient agar plate that has been precoated with bacteria. After the fluid has been absorbed, seal the plates with a 5- to 6-in. length of 1-in.-wide Parafilm strip. Incubate the plate inverted at 37°C in room air. Using a wax pencil or laboratory marker, you may want to make a circle on the underside of the plate to indicate exactly where the specimen was inoculated onto the agar.
B. For tissue samples, triturate a small piece of the tissue (brain, lung, skin abscess, corneal biopsy, or similar specimens) in a small quantity (ca. 0.5 ml) of ameba saline. Process as above. Corneal smear, ear discharge material, etc., may be placed directly on the agar surface. Incubate central nervous system tissues at 37°C (room air) and tissues from other sites at 30°C.
C. Water samples of 10 to 100 ml may be processed to isolate these amebae. First, filter the water sample through three layers of sterile gauze or cheesecloth to remove leaves, dirt, etc. Next, either (i) filter the sample through a sterile 5.0-µm cellulose acetate membrane (47 mm in diameter), invert the membrane over a nonnutrient agar plate precoated with bacteria, seal, and incubate the plates as above; or (ii) centrifuge the water sample for 10 min at 250 × g. Aspirate the supernatant, suspend the sediment in about 0.5 ml of ameba saline, and deposit this suspension in the center of the nonnutrient agar plate precoated with bacteria. Seal and incubate the plate at 37°C as before.
D. For soil samples, mix about 1 g of the soil sample with enough ameba saline (ca. 0.5 to 1 ml) to make a thick slurry. Inoculate this slurry in the center of the nonnutrient agar plate precoated with bacteria, and incubate as above.
E. For contact lens solutions, small volumes (ca. 1 to 2 ml) may be inoculated directly onto the nonnutrient agar plates precoated with bacteria. Larger volumes (2 to 50 ml) should be centrifuged as in step 3, and the sediment should be inoculated onto the center of the nonnutrient agar plate and incubated as above.
4.