A Multiplex PCR Assay for Molecular Recognition of T. gondii Stage-Specific Genes

Toxoplasmic encephalitis (TE) is one the most frequent opportunistic infections in immunocompromised patients including those with AIDS. Disease in these persons is more commonly due to reactivation of chronic infection than to newly acquired infection. One important event underlying TE is the stage conversion between the encysted bradyzoites and the tachyzoites [7–8,13]. The diagnostic investigation of stage conversion is not easy at serological level [12]. In recent years, advanced improvement in T. gondii diagnosis has been achieved with the development of polymerase chain reactions (PCR)-based techniques, which detect the parasite's DNA in clinical specimens. Most of these assays make use of the multi-copy Bl gene of T. gondii, which is well conserved among different parasite strains [1]. But this does not distinguish DNA of bradyzoites from DNA of tachyzoites, especially during latent infection or when specific treatment has been started [4–6]. The ability to recognize individual stages by detecting specific genes would thus be crucial for correctly diagnosing TE. In the present study we carried out a novel “in house” multiplex nested (n) PCR and reverse transcriptase (RT)-PCR method as a diagnostic tool for detecting and differentiating tachyzoite and bradyzoite-specific gene targets simultaneously in cerebrospinal fluid (CSF) specimens from patients with TE.

MATERIALS AND METHODS

CSFs were collected from 19 immunocompromised patients (AIDS, cancers, transplants) with TE that were hospitalized in different Italian Institutions: Ferrara, (Section of Infectious Diseases, and Haematology, University of Ferrara) and Rome (Infectious and Tropical Diseases, Policlinico Umberto 1*, University “La Sapienza”). TE cases were separated into: a) 1^st episodes from whom CSF samples were taken before starting specific anti-T. gondii therapy (n = 8), and b) relapse, most of whom were receiving maintenance anti-Toxoplasma prophylaxis after resolution of an acute episode of TE (n = 11). None of these patients had received H AART at the time of sample collection.

The specific primers used for the respective PCR programs are shown in Table 1. CSF specimens were amplified using n-PCR by the currently used stage-specific pair of primers, which amplify genes coding for the tachyzoite surface specific proteins (SAG1), the bradyzoite 65 kDa surface proteins (MAGI) or the 18 kDa protein (SAG4), or those in both stages (Bl) of T. gondii [9–11,1]. Five μl of CSF DNA were used in 50 μ of PCR reaction mixture containing 2 U Taq DNA polymerase, 0.2 mM dNTP, 1.5 mM MgCl₂, and 50 pmol of each pair of outer primers for SAGl or MAGI and SAG4 genes. After denaturation at 95°C for 10 min, PCR conditions were modified with respect to those previously reported by employing a novel “touchdown” amplification program [3]. This program consisted of a first round of amplification using 28 cycles of 30 s at 95°C, 30 s at 62°C minus 0.5°C per cycle, and 50 s at 72°C, plus additional 15 cycles of 30 s at 95°C, 30 s at 48°C, and 50 s at 72°C. The PCR products were diluted 1:10, and 5 ml were amplified by n-PCR with inner primers under the same conditions used for the first round of amplification. Multiplex RT-PCR was done in single tubes by the Access RT-PCR System (Promega). The first cycle of 45 s at 48°C and 2 min at 94°C was used for the first strand cDNA, and was followed by two rounds of the cycling program described above for the “touchdown” n-PCR. 50 pmol each of the downstream and upstream outer primers were used for the SAG1, SAG4, MAGI and Bl genes. Specificity of the oligonucleotides was determined by PCR amplification of DNA extracted from a number of bacteria, fungi (Aspergillus, Cryptococcus), virus (CMV) and from healthy humans. Also, a theoretical alignment from the GenBank(tm) BLAST (Basic Local Alignment Search Tool) was employed. Southern blot analysis using gene-specific probes in a chemiluminescent procedure (Amersham, International plc, England) was used to confirm the PCR results.

RESULTS AND CONCLUSIONS

Table 2 summarizes the principal findings obtained with the n- PCR and RT PCR techniques. In the samples from patients with TE relapses, oligonucleotides targeting SAG4 and MAGI genes were more sensitive in detecting T. gondii-specific DNA compared to samples from TE-l^st episode patients (75% vs. 25%; p < 0.01). However, no differences in detection rates were observed between patients with relapses taking anti-TE maintenance prophylaxis or full dose regimens.

In both study groups, the n-PCR results were consistent with those of RT-PCR. This technique was shown to be more sensitive in patients with relapse compared to 1^st episode (p<0.01). With the Bl gene, positive results were obtained in 5 TE patients with 1^st episode who had undergone lumbar puncture before starting specific treatment, while only 1 positive case was found among patients with relapse (62.5% vs. 9.09%; p<0.001). Since MAG-1 gene transcripts are expressed more in bradyzoites than in tachyzoites and are principally regulated during conversion from the tachyzoite to bradyzoite stage, RT-PCR assay for MAG-1, in parallel with SAG-4, could be more useful than other bradyzoite-specific mRNA markers in detecting the bradyzoite conversion in clinical investigations [9]. The SAG1 gene-specific primers also detected more relapse cases compared to TE 1^st episode cases, but these differences were not significant.

These preliminary results confirm previous findings in studies employing bradyzoite-specific oligonucleotides to detect T. gondii in patients with TE relapses [2]. Simultaneous gene amplification by multiplex PCR could be an efficient, sensitive, and specific method for laboratory diagnosis of T. gondii reactivation.

AKNOWLEDGMENTS

Work supported in part by grants from Italian Ministry of University and Scientific Research (MURST- ex 40%, 2001 and ex 60%, 2001).