Multiplex RT-PCR Assay for the Detection of stage conversion processes in Toxoplasma gondii: A Toxoplasmic Encephalits

Abstract

The protozoan parasite, Toxoplasma gondii, is capable of establishing a chronic infection in its hosts by differentiating from rapidly dividing tachyzoites into dormant bradyzoites. The stage conversion process from bradyzoite back into tachyzoite is a major cause of Toxoplasmic encephalitis (TE) in acquired immune deficiency syndrome (AIDS) patients. Until now, the diagnosis of TE remains extremely difficult and mainly relies on clinical and radiological features or on the improvement of clinical symptoms after specific treatment. Here, we have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that facilitates the analysis of large numbers of samples for differences in sag-1 and sag-4 mRNA abundance, genes which are selectively expressed by either tachyzoites or bradyzoites. In our assay, we furher coamplified alpha-tubulin cDNA as an internal control. The results obtained from our RT-PCR assay show that the multiplex RT-PCR consisting of the parallel amplification of sag-1, sag-4 and tubulin cDNA offers a simple, rapid and sensitive technique for the detection of T. gondii tachyzoite/bradyzoite stage conversion. The established RT-PCR technique may serve in the future as a feasible alternative tool to diagnose TE in severely immune-compromised patients.