The role of stage-specific oligonucleotide primers in providing effective laboratory support for the molecular diagnosis of reactivated Toxoplasma gondii encephalitis in patients with AIDS

Abstract

The switch from bradyzoites to tachyzoites is the fundamental pathogenic event that leads to Toxoplasma gondii encephalitis (TE) in patients with AIDS. Distinction between these stages is difficult, particularly when specific treatment has been started. A new approach consisting of a nested PCR (n-PCR) assay was performed on cerebrospinal fluid (CSF) specimens collected from AIDS patients with TE before or after antiparasitic therapy was initiated, to assess the efficacy of primer sets which amplify target sequences expressed on bradyzoites (SAG4 and MAG1), tachyzoites (SAG1) or both stages (B1) of T. gondii. CSF specimens were obtained from 46 patients with AIDS, of whom 27 had TE (16 first episode, 11 relapse) and 19 had other AIDS-related brain lesions (AIDS-OBL) in the absence of TE. CSF specimens from 26 HIV-negative and immunocompetent patients were also checked. All samples were tested with different primer pairs targeting the B1, SAG-1, SAG-4 and MAG-1 genes. With B1, 75% of patients with first episodes of TE were positive, compared with 36.3% of those with relapse of TE and 5.2% of those with AIDS-OLB. The SAG1 gene yielded positive values in 28.7% and 45.4% of patients with first episodes of TE or relapse of TE, respectively, and in none of the controls. With the SAG4 and MAG1 genes, 72.7% of patients with relapse of TE were detected, compared with 25% of patients with first episodes of TE and 5.2% with AIDS-OLB. None of the HIV-negative subjects showed positive PCR reactions. These results demonstrate that specific primers for the genes SAG4, MAG1 and SAG1 may be useful in AIDS patients with relapse of TE, in whom the use of PCR targeting the B1 gene may fail to detect DNA, especially when prophylaxis or treatment has been started.

Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in human peripheral blood

“…PCR has been used to detect Toxoplasma infection in several body fluids, including blood, representing a noninvasive alternative to diagnose the infection (Bastien 2002;Contini et al 2002;Montoya 2002;Martino et al 2005). Previous report from our group has shown that the usual B1 gene amplification may show false positive results and a hybridization step is mandatory to reach a final and specific result (Kompalic-Cristo et al 2004).…”

Molecular Methods for Diagnosis of Viral Encephalitis

“…Specific primers for the genes SAG4, MAG1, and SAG1 may be useful in AIDS patients with relapse of TE, in whom the use of PCR targeting of the B1 gene may fail to detect DNA, especially when prophylaxis or treatment has been started (58,62). These assays may aid in distinguishing asymptomatic latent infection from reactivated disease in these patients.…”

Antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus-infected patients

“…Although parasite DNA detection by the polymerase chain reaction (PCR) has been reported as a rapid and effective technique in the early diagnosis of toxoplasmosis in HIV-infected patients, [19][20][21] its sensitivity and specificity are variable. Moreover, lack of expertise and equipment in most diagnostic centres in developing countries poses problems in its use, and antigen detection may prove a better option in such circumstances.…”