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 for the culture of Plasmodium falciparum were described in 1976 (42) and have been improved and modified since that time (43, 44). Life cycle stages of the five Plasmodium spp. that infect humans have been established in vitro. Of these five, P. falciparum and P. knowlesi are the only species for which all stages have been cultured in vitro. The life cycle includes the exoerythrocytic stage (within liver cells), the erythrocytic stage (within erythrocytes or precursor reticulocytes), and the sporogonic stage (within the vector). Culture media generally consist of a basic tissue culture medium to which serum and erythrocytes are added. Most of the culture methods have been directed toward the stage found in the erythrocyte. This stage has been cultivated in petri dishes or other containers in a candle jar to generate elevated CO2 levels or in a more controlled CO2 atmosphere. Later developments employed continuous-flow systems to reduce the labor-intensive requirement for replenishing the system with fresh media. The exoerythrocytic and sporogonic life cycle stages have also been cultivated in vitro. Although cultivation is of great help in understanding the biology of Plasmodium, it does not lend itself to use for routine diagnostic purposes (7).
The availability of the microaerophilous stationary phase (MASP) culture technique, in which the parasites proliferate in a settled layer of blood cells, has provided an opportunity to study Babesia, a formerly obscure disease agent regarded as within the purview of veterinary parasitology, in the laboratory. A number of Babesia spp. have been established in continuous culture using the MASP technique. It is possible to study the basic biology of the organism—as well as host-microbe interactions, immune factors triggered by the parasite, factors involved in innate resistance of young animals to infection, and antimicrobial susceptibility—to a degree not possible before the availability of cultures. These culture systems can produce quantities of parasite nucleic acid needed for defining phylogenetic relationships, developing diagnostic methods for parasite detection in asymptomatic individuals, and producing parasite antigens and attenuated strains of Babesia that could be used for immunization (5).
The in vitro cultivation of Cryptosporidium has improved significantly in recent years. These obligate intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerator temperatures (4 to 8°C). While continuous culture and oocyst production have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields tend to be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation (1). Recent studies indicate that primary human intestinal epithelial cells (PECs) support Cryptosporidium better than other existing cell lines (45).
Although various microsporidia that infect humans can be identified from clinical specimens by serologic and/or molecular methods, none of these methods are commercially available. Unfortunately, in some cases microscopic examination of biopsy specimens does not yield conclusive results. It is also possible that microsporidial organisms may be present in very small numbers, which can be easily missed during routine histologic examinations. Some microsporidia such as Encephalitozoon, Enterocytozoon,