Direct Detection of Toxoplasma gondii from Clinical Specimens

The protozoan Toxoplasma gondii is estimated to be carried by one third of the world's population with infection typically occurring by eating infected meat, contact with a cat that has itself recently been infected, or by transmission from mother to fetus. During acute toxoplasmosis, symptoms are often influenza-like: swollen lymph nodes, or muscle aches and pains that last for a month or more. Rarely, a patient with a fully functioning immune system may develop eye damage from toxoplasmosis.

Toxoplasmosis can be difficult to distinguish from that of primary central nervous system lymphoma, and as a result, the diagnosis is made by a trial of therapy (pyrimethamine, sulfadiazine + leucovorin), or a brain biopsy if the drugs produce no effect. Serologic testing for both IgG and IgM can determine if and when individual is/was infected. Additionally, PCR has been successfully used to diagnose congenital, ocular, cerebral and disseminated toxoplasmosis. PCR performed on amniotic fluid has revolutionized the diagnosis of fetal T. gondii infection by enabling an early diagnosis to be made, thereby avoiding the use of more invasive procedures on the fetus. PCR has allowed detection of T. gondii DNA in brain tissue, CSF, vitreous and aqueous fluid, BAL, urine, amniotic fluid and peripheral blood.

Among PCR assays, the B1 gene is consistently determined to be a useful target. B1 is a tandem-arrayed 35-fold-repetitive gene which has been used for both detection and typing of Toxoplasma strains in clinical samples. The UWMC Molecular Diagnosis laboratory utilizes a 2-step, hemi-nested PCR approach to amplify a region of the B1 gene. Identification of Toxoplasma gondii depends on SYBR-green melting curve and evaluation of the sequenced product.