href="#ulink_ed58e1ed-33c5-590e-a1a0-9e8da80ea028">16–19). Daily search for furrows on agar plates for up to six consecutive days results in increased sensitivity for diagnosis of both S. stercoralis and hookworm infections. Also, a careful search for S. stercoralis should be performed for all patients with comparable clinical findings before a diagnosis of idiopathic eosinophilic colitis is made, because the consequent steroid treatment may have a fatal outcome by inducing widespread dissemination of the parasite (24). Human T-cell leukemia virus type 1 (HTLV-1) infection is endemic in a number of Latin American countries. HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma are emerging diseases in the region. S. stercoralis hyperinfection syndrome and therapeutic failure in apparently healthy patients with nondisseminated strongyloidiasis may be markers of HTLV-1 infection (25).
Stool is placed onto agar plates, and the plates are sealed to prevent accidental infections and held for 2 days at room temperature. As the larvae crawl over the agar, they carry bacteria with them, thus creating visible tracks over the agar (Fig. 4.4). The plates are examined under the microscope for confirmation of the presence of larvae, the surface of the agar is then washed with 10% formalin, and final confirmation of larval identification is made via wet examination of the sediment from the formalin washings (Fig. 4.5).
Figure 4.4 Agar plate culture method for Strongyloides stercoralis. (Upper) Agar plate showing bacterial growth after being distributed over the plate by the movement of the larval worms. (Lower) More random pattern of bacterial growth from the inoculation of bacteria over the agar from the movement of the larval worms (image courtesy of Audrey N. Schuetz, Weill Cornell Medical College). doi:10.1128/9781555819002.ch4.f4
Figure 4.5 Agar culture method for Strongyloides stercoralis. (1) Agar plates are prepared. (2) Agar is dried for 4 to 5 days on the bench top. (3) Plates are stored in plastic bags. (4) Fresh stool is submitted to the laboratory. (5) Approximately 2 g of stool is placed onto an agar plate. (6) The plate is sealed with tape. (7) The culture plate is incubated at 26 to 33°C for 2 days. (8) The plate is examined microscopically for the presence of tracks (bacteria carried over agar by migrating larvae). (9) 10% formalin is placed onto agar through a hole made in the plastic with hot forceps. (10) Material from the agar plate is centrifuged. (11) The material is examined as a wet preparation for rhabditiform or filariform larvae (high dry power; magnification, ×400). (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch4.f5
Occasionally, finding nematode larvae in sputum or bronchoalveolar lavage fluid specimens may be very suggestive of a potential infection with S. stercoralis. In Fig. 4.6, larvae can be seen stained with Giemsa stain or a Gram stain. Once larvae are seen in respiratory specimens, fecal specimens can be collected for agar plate cultures to confirm strongyloidiasis.
Figure 4.6 Strongyloides stercoralis larvae. (Upper) Giemsa-stained larvae in a specimen of bronchoalveolar lavage fluid (courtesy of Marc Long). (Middle) Gram-stained larva in thoracic fluid. (Lower) Gram-stained larva in a respiratory specimen (sputum). doi:10.1128/9781555819002.ch4.f6
Agar
1.5% agar
0.5% meat extract
1.0% peptone
0.5% NaCl
Note Positive tracking on agar plates has been seen on a number of different types of agar. However, the most appropriate agar formula is that listed above.
Quality Control for Agar Plate Culture for Strongyloides stercoralis
1. Follow routine procedures for optimal collection and handling of fresh fecal specimens for parasitologic examination.
2. Examine agar plates to ensure that there is no cracking and the agar pour is sufficient to prevent drying. Also, make sure that there is no excess water on the surface of the plates.
3. Review larval diagrams and descriptions for confirmation of larval identification.
4. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors for all objectives should be posted on the microscope for easy access (multiplication factors can be pasted on the body of the microscope).
5. Record all QC results (condition of agar plates).
Procedure for Agar Plate Culture for Strongyloides stercoralis
1. Place approximately 2 g of fresh stool in the center of the agar plate (area approximately 1 in. in diameter).
2. Replace the lid, and seal the plate with cellulose tape.
3. Maintain the agar plate (right side up) at room temperature for 2 days.
4. After 2 days, examine the sealed plates through the plastic lid under the microscope for microscopic colonies that develop as random tracks on the agar and evidence of larvae at the ends of the tracks away from the stool.
Note It has been documented that daily search for tracks on agar plates for up to six consecutive days results in increased sensitivity for diagnosis of both S. stercoralis and hookworm infections (17). When trying to rule out strongyloidiasis in immunocompromised patients or in those who may receive immunosuppressive drugs, it is recommended that two plates be set up, one that can be examined after 2 days and one that can be examined after the full 6 days.
5. With the ends of hot forceps, make a hole in the top of the plastic petri dish.
6. Gently add 10 ml of 10% formalin through the hole onto the agar surface, and swirl to cover the surface and rinse the agar plate. Allow to stand for 30 min.
7. Remove the tape and lid of the agar plate. Pour the 10% formalin through a funnel into a centrifuge tube. Do not try and pour the formalin off directly into the centrifuge tube—the tube opening is too small, and formalin will be spilled onto the counter.
8. Centrifuge the formalin rinse fluid 5 min at 500 × g.
9. Prepare a wet smear preparation from the sediment and examine with a 10× objective (low power) for presence of larvae. If larvae are found, confirm the identification with a 40× objective (high dry power).
Results and Patient Reports from Agar Plate Culture for Strongyloides stercoralis
Larval nematodes of hookworm, S. stercoralis, or Trichostrongylus spp. may be recovered. If Strongyloides organisms are present, free-living stages and larvae may be found after several days on the agar plates.
1. Report “No larvae detected” if no larvae could be detected at the end of incubation and rinse procedure.
2. Report larvae detected by agar plate
