Isolation of Toxoplasma gondii

Maija Lappalainen

Culture of Toxoplasma gondii is a specific method giving an unequivocal proof of infection. Parasite isolation or identification is useful, especially in the diagnosis of foetal infection or in immunocompromised patients.

Isolation is usually performed by inoculation of suspect material into white mice or cell cultures. Toxoplasma gondii can multiply in a variety of cell lines, such as VERO, MRC-5, Hep-2, L-cells, and primary monkey kidney. The human embryonic fibroblast line MRC-5 is widely used to culture Toxoplasma from clinical samples. Toxoplasma gondii has been isolated in various tissues and body fluids: blood, placenta, brain, skeletal muscle, lung biopsy, heart, cerebrospinal fluid, subretinal fluid, aqueous humor, amniotic fluid, and broncho-alveolar lavage fluid (Ho-Yen and Joss 1992). Isolation from tissues may reflect only the presence of tissue cysts and does not necessarily mean active acute infection. From lymph nodes, however, positive isolation probably indicates the presence of tachyzoites, because cysts are rarely found in nodes.

Specimens should be injected into animals and cell cultures as soon as possible after collection to prevent death of the parasite. Sample preparation before inoculation is usually needed (Table 30.1). Formalin kills the parasite, and freezing may result in the death of both the tachyzoite and the cyst forms in tissues ( James et al. 1996). If short-term storage of specimens is necessary, refrigeration at +4°C is preferred. This maintains the encysted form for up to two months (if kept moist) and prevents death of the tachyzoite for several days. Toxoplasma gondii survive in blood for a week or even longer.

Table 30.1 Sample preparation for isolation of Toxoplasma gondii

Specimen

Management before inoculation

Inoculuma

Blood Body fluids

Centrifugation Centrifugation or none

Clot

Cells or as such

Sediment

Solid tissues (muscle, placenta, etc.)

Mincing followed by trypsinb digestion. Filtration, centrifugation. Sediment is washed and resuspended in sterile saline

Notes a Inoculum volume is usually 0.5-1.0ml.

b 0.25% trypsin treatment for 1-2h at +37°C, if large amounts of tissues are prepared.

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