Development of Sensitive Nested Real-time PCR for Diagnosis of Acute and Chronic Phases of Toxoplasmosis in Mice Model

Abstract

Toxoplasma gondii is an intracellular parasite that causes a variety of clinical manifestations. Acute and chronic phases of toxoplasmosis are considered as the presence of actively proliferating tachyzoites in the nucleated cells of mammalian hosts such as humans, and spread through blood to other parts of the body, which subsequently forms tissue cysts. The present study was aimed to evaluate the diagnostic value of nested real-time polymerase chain reaction (PCR) for the acute and chronic phases of toxoplasmosis in the laboratory mice using compared to conventional real-time PCR. To induce acute toxoplasmosis, 103 tachyzoites of Toxoplasma gondii RH strain were intraperitoneally inoculated to 25 BALB/c mice. In order to induce chronic toxoplasmosis, the mice were subcutaneously infected by the parasite and then treated with sulfadiazine from day one to day 14 post-injection. Genomic DNA was extracted from blood and brain tissues. Real-time and nested real-time PCR targeting 529 bp repeated element (RE) was performed. All mice with acute infection were positive for Toxoplasma gondii using nested real-time PCR and 21 were positive by real-time PCR. In the chronic phase, all blood samples were negative with real-time PCR and three were positive using nested real-time PCR. However, of the 25 brain samples, 28%, 52% and 72% were positive with the microscopic, real-time PCR and nested real-time PCR methods, respectively. The results of the present study showed that the molecular methods have high sensitivity for the diagnosis of acute toxoplasmosis. In the chronic phase, a blood sample is not suitable for the detection of the infection, and other tissue samples may be used instead. Also, nested real-time PCR has even higher sensitivity compared to the conventional real-time PCR.