Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction

TLDR

The maximal cellular infiltration in cerebrospinal fluid of patients with toxoplasmic encephalitis is about 10^5 cells/ml. We used PCR targeting the highly repetitive B1 gene to detect Toxoplasma gondii. The assay amplified the B1 gene from a single parasite in crude lysate, detected as few as 10 parasites among 100,000 leukocytes, was positive in all six tested strains, showed no cross‑reactivity with other CNS organisms, and thus provides highly sensitive and specific diagnosis of toxoplasmosis in immunocompromised patients and fetuses.

Abstract

We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.