Efficacy of a novel reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting Toxoplasma gondii bradyzoite gene expression in human clinical specimens.

Abstract

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay, was performed to evaluate the transcription degree of bradyzoite- or tachyzoite-specific genes of Toxoplasma gondii on cerebrospinal fluid (CSF) specimens from AIDS patients with toxoplasmic encephalitis (TE), and to distinguish an asymptomatic latent infection from a reactivated disease. This method was compared with nested DNA amplification (n)-PCR. The mRNA expression of the representative T. gondii cystic matrix (MAG1) or bradyzoite-specific (SAG4) genes was investigated on CSF obtained from AIDS patients with first episode (no. 11) or relapse (no. 8) of TE. The mRNA expression of tachyzoite-specific (SAG1) gene was also studied. New designed oligonucleotide primers and probes, which identify a 212 bp fragment inside to the open reading MAG1 sequence, were employed in both RT-PCR and n-PCR assays. Oligo-dT primed cDNA synthesis appeared a suitable method for subsequent analysis by n-PCR. RT-PCR has been shown to be more sensitive and specific than n-PCR. MAG1 and SAG4 gene expression was detected in 8 (100%) and 6 (75%) patients with TE relapses, respectively, while SAG1 detected 7 (63%) patients with TE first episode. These findings suggest that RT-PCR method is able to identify the bradyzoite stage of T. gondii especially in patients who are at risk for TE relapse.