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. Using the low-power (10×) objective, examine the plates microscopically for amebae (cysts or trophozoites) every day for 10 days. Thin linear tracks (areas where amebae have ingested bacteria) might also be seen. If amebae are seen, circle that area with a wax pencil, carefully remove the Parafilm seal in a biological safety cabinet, open the lid of the petri dish, and carefully cut out the marked area from the agar with a spatula that has been heated to red hot and cooled before use to prevent contamination. Transfer the piece face down onto the surface of a fresh agar plate coated with bacteria, seal the plate with Parafilm, and incubate as before. Naegleria and Acanthamoeba spp. can be cultured by this method and, with periodic transfers, can be maintained in the laboratory indefinitely. In lieu of subcultures, the organisms can also be frozen for long-term storage. Under the microscope, the amebae resemble small uneven spots; observation for several seconds may reveal organism motility. After 4 to 5 days of incubation, the amebae begin to encyst and both trophozoites and cysts are visible. Unfortunately, Balamuthia spp. cannot be grown by using this system; they can be grown by using tissue culture methods on monkey kidney or lung fibroblast cell lines.
5. The enflagellation experiment is carried out as follows.
A. Examine the plates every day for signs of amebae. If present, amebae will feed on bacteria, multiply, and cover the entire surface of the plate within a few days. Once the food supply is exhausted, the amebae will differentiate into cysts.
B. Use a wax pencil to mark the area containing a large number of amebic trophozoites.
C. Using a bacteriological loop, scrape the surface of the agar at the marked area and transfer several loopfuls of the scraping to a sterile tube containing about 2 ml of sterile distilled water. Alternately, flood the surface of the agar plate with about 10 ml of sterile distilled water, gently scrape the agar surface with a loop, transfer the liquid to a sterile tube, and incubate at 37°C.
E. Periodically examine the tube with an inverted microscope for the presence of flagellates.
a. N. fowleri, the causal agent of primary
