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 amebic meningoencephalitis, undergoes transformation to a pear-shaped flagellate, usually with two flagella but occasionally with three or four flagella; the flagellate stage is a temporary nonfeeding stage and usually reverts to the trophozoite stage (Fig. 8.2). N. fowleri trophozoites are typically ameba-like and move in a sinuous way. They are characterized by a nucleus with a centrally located, large nucleolus. The trophozoites are also characterized by the presence of a contractile vacuole that appears once every 45 to 50 s and discharges its contents. The contractile vacuole looks like a hole or a dark depression inside the trophozoite and can be easily seen when examining the plate under the 10× or 40× objective. When the food supply is exhausted, N. fowleri trophozoites differentiate into spherical, smooth-walled cysts.
b. In contrast, Acanthamoeba spp., which cause keratitis and granulomatous amebic encephalitis, do not transform into the flagellate stage. Acanthamoeba trophozoites are characterized by the presence of fine, thorn-like processes that are constantly extended and retracted. The trophozoites produce double-walled cysts characterized by a wrinkled outer wall (ectocyst) and a polygonal, stellate, oval, or even round inner wall (endocyst). The trophozoites are also characterized by the presence of the contractile vacuole, which disappears and reappears at regular intervals (45 to 50 s).
c. The cysts of both Acanthamoeba and Naegleria spp. are uninucleate.
d. Immunofluorescence and immunoperoxidase tests with monoclonal or polyclonal antibodies (available at the Centers for Disease Control and Prevention) are helpful in differentiating Acanthamoeba and Balamuthia spp. in fixed tissue.
Figure 8.2 Naegleria fowleri flagellate stage. (Upper) When placed in distilled water (enflagellation test), N. fowleri, the causal agent of primary amebic meningoencephalitis, undergoes transformation to a pear-shaped flagellate, usually with two flagella but occasionally with three or four flagella; the flagellate stage is a temporary nonfeeding stage and usually reverts to the trophozoite stage. (Lower) Naegleria flagellate stage (microtubules are highlighted in green, basal bodies in red, and DNA is stained blue). (Courtesy of Lillian Fritz-Laylin; from http://genome.jgi-psf.org/Naegr1/Naegr1.home.html.) doi:10.1128/9781555819002.ch8.f2
Quality Control for Pathogenic Free-Living Amebae
The following control strains should be available when using these cultures for clinical specimens: ATCC 30010 (Acanthamoeba castellanii) and ATCC 30215 (N. fowleri). ATCC strains of E. coli or E. aerogenes are not necessary. Any routine clinical isolate or stock organism is acceptable.
1. Check all reagents and media (ameba saline, distilled water, and nonnutrient agar plates) each time they are used or periodically (once a week).
A. The media and all solutions should be free of any visible signs of precipitation and bacterial and/or fungal contamination.
B. Examine the nonnutrient agar plates under the 4× objective of an inverted or binocular microscope, and make sure that no fungal contamination has occurred.
2. Maintain stock cultures of A. castellanii and N. fowleri at 25°C.
A. Transfer stock cultures monthly, using nonnutrient agar plates prepared with Page’s ameba saline and the bacterial overlay of E. coli.
B. N. fowleri measures 10 to 35 µm and demonstrates an eruptive locomotion by producing smooth hemispherical bulges. The cyst produces smooth (7- to 15-µm) walls. The flagellate stage does not have a cytostome.
C. A. castellanii is 15 to 45 µm and produces fine, tapering, hyaline projections called acanthapodia. It has no flagellate stage but produces a double-walled cyst with a wrinkled outer wall (10 to 25 µm).
D. Trophozoites of Naegleria and Acanthamoeba spp. are uninucleate, characterized by a large, dense central nucleolus.
E. For staining, a slide prepared from a stock strain of amebae is run in parallel with the patient slide. Staining results are acceptable when the control amebae stain well.
F. Plate both stock cultures onto fresh media, and incubate at 37°C parallel with the patient culture. Culture results are acceptable when growth appears by day 7.
G. Run N. fowleri in parallel with the patient culture being observed for enflagellation. The test results are acceptable when free-swimming, pear-shaped flagellates with two flagella are observed in 2 to 24 h on the control slide.
3. Depending on use, the microscope(s) may have 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 For patient specimens, if a plate is positive for amebae and the amebae transform into flagellates, the specimen should be reported as positive for N. fowleri. If the amebae do not transform into flagellates even after overnight incubation, if the trophozoites possess the characteristic acanthapodia, if they show a large centrally placed nucleolus in the nucleus on trichrome stain, and if the trophozoites differentiate into the characteristic double-walled cysts, the specimen should be reported as positive for Acanthamoeba sp. For contact lens solution, if the plates are positive for amebae and the amebae do not transform into flagellates but differentiate into cysts with a wrinkled outer ectocyst and an inner stellate, polygonal, oval, or round endocyst, the specimen should be reported as positive for Acanthamoeba sp. Naegleria spp. have not been isolated from contact lens solutions; however, small amebae (e.g., hartmannellid or vahlkampfiid amebae, which produce smooth-walled cysts), probably contaminants, have occasionally been isolated from these solutions. Plates inoculated with water samples are usually positive for many genera and species of small free-living amebae (freshwater is their normal habitat). Therefore, the sample should be reported as positive for small free-living amebae. The physician should be notified immediately if patient specimens are positive for Acanthamoeba or Naegleria spp.
Most patient specimens, especially