material from skin lesions from cutaneous leishmaniasis patients; bone marrow aspirates or splenic aspirates from visceral leishmaniasis patients; or normal skin biopsy specimens, lymph node aspirates, or pieces of liver and spleen from suspected or potential wild- or domestic-animal reservoirs) into the culture tubes. For Chagas’ disease, inoculate a few drops of buffy coat into the culture tubes.
3. Add 0.5 ml of overlay (either saline or other overlay, depending on the medium). The organisms will develop in the fluid condensate and overlay at the bottom of the slant (Fig. 8.5).
4. Incubate the tubes at 20 to 24°C.
5. Once every 2 to 3 days, remove a drop of medium and examine it under the low power (100×) of a microscope, preferably one equipped with phase-contrast optics.
6. If promastigotes are seen, inoculate a couple of drops of the medium into fresh culture tubes. Add a couple of drops of 0.85% saline or the overlay solution (depending on the culture medium used) to the old tube.
7. If visible contamination occurs, add antibiotics to the overlay (to contain 200 U of penicillin/ml and 200 µg of streptomycin/ml). The parasites will not proliferate if bacterial contamination is present.
8. Incubate the tubes containing the patient specimen for at least 2 weeks at 28°C or room temperature.
9. If no organisms are seen even after 2 weeks of incubation, examine several drops of fluid under the microscope for promastigotes. The culture should be observed for 1 month before being signed out as negative. Transfers to fresh medium should be made once or twice a week after the culture is established.
Quality Control for Flagellates of Blood and Tissue
The following control strains should be available when using these cultures for clinical specimens: ATCC 30883 (Leishmania mexicana) and ATCC 30160 (T. cruzi).
1. Check all reagents and media at least once a week. The media should be free of any signs of precipitation and bacterial and/or fungal contamination.
2. The microscope(s) should have been 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.
3. Maintain stock cultures of Leishmania spp.
A. Transfer stock cultures weekly.
a. Stock organisms should always be cultured at the same time a patient specimen is inoculated into culture medium.
b. If the stock organisms multiply and remain viable during the 96 h, patient results can be reported.
B. Stain
a. Stain a slide prepared from stock culture in parallel with the patient slide.
b. Staining results are acceptable when the control organisms stain well.
Note Cultivating the organism from suspected materials provides a definitive diagnosis, but it may take as many as 3 to 7 days. Every effort, therefore, must be made to microscopically examine wet smears and/or permanent stained smears so that appropriate therapy can be instituted without delay if findings are positive (Fig. 8.6) (37, 38).
Figure 8.6 Leishmania spp. from culture systems. (Upper left) Stained smear of culture fluid sediment [U3] showing promastigotes of Leishmania sp. (Upper right) Stained smear of culture fluid sediment showing promastigotes of Leishmania sp. (higher magnification). (Lower left) Stained Leishmania promastigote. (Lower right) Leishmania major promastigotes during infection of primary fibroblast culture. Cells are stained with antitubulin (green) and antiactin (red). (Courtesy of the Pasteur Institute, Molecular Parasitology and Signaling Image Bank). doi:10.1128/9781555819002.ch8.f6
The diagnosis of toxoplasmosis may be difficult, because the clinical symptoms mimic a number of various infectious and noninfectious diseases. Serologic tests that are often used for diagnosis may be insensitive in patients lacking normal immune responses. Sometimes even examination of histologic material does not reveal the organisms. With the increase in the number of laboratories using tissue culture techniques for viral pathogens, these techniques have been used for the isolation and identification of T. gondii. The following procedure has been recommended for biopsy specimens, brain, liver, spleen tissue, CSF, amniotic fluid, and buffy coat preparations, and they may be particularly helpful in making the diagnosis in immunosuppressed patients (39). The procedure for buffy coat cells is as follows.
1. Collect 10 ml of blood anticoagulated with preservative-free heparin.
2. Allow the blood to sediment via gravity.
3. Remove the buffy coat (by an aseptic technique), and separate the cells from the plasma by centrifugation at 800 × g for 10 min.
4. Wash the buffy coat cells three times with Eagle’s minimal essential medium (GIBCO).
5. Inoculate the washed buffy coat material onto complete human foreskin fibroblast (HFF) monolayers (in tubes and shell vials). One HFF tube and two HFF vials should be inoculated for each patient specimen.
6. Observe the cultures weekly for cytopathic effect.
7. The shell vial coverslips can be fixed and stained at 7 and 14 days postinoculation for an indirect fluorescent-antibody (IFA) assay and observed for the tachyzoites.
Note CSF, placental tissue, or other tissues can also be used to inoculate tissue culture monolayers. Uncentrifuged CSF (0.1 to 1 ml) can be used. If more than 1 ml is submitted, the specimen should be centrifuged for 10 min at 500 × g. Positive and negative controls must be tested with each set of patient specimen vials.
Three continuous cell lines (HeLa, LLC, and Vero) and three cell culture methods (culture in conventional flasks, culture in membrane-based flasks, and an automated culture system) were investigated (Fig. 8.7). Overall, HeLa was the cell line of choice. Continuous passage in flasks was successful, and HeLa-derived tachyzoites can be used for the dye test, if applicable in your laboratory setting (40). Another study indicates that THP1 cells serve as a good model of invasion for T. gondii (41).
Figure 8.7 Toxoplasma gondii RH tachyzoites replicated in Vero cell cultures. A rosette of many tachyzoites is seen at the left and several parasite pairs are at right (arrows). (Courtesy of I. Canedo-Solares, Abstract 42.009, 15th International Congress on Infectious Diseases, Bangkok, Thailand, 2012). doi:10.1128/9781555819002.ch8.f7
Techniques
