Efficacy of a novel reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting Toxoplasma gondii bradyzoite gene expression in human clinical specimens.
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.
Cultrera
R
R
Infectious Diseases, Department of Clinical and Experimental Medicine, University of Ferrara, Italy. ctr@unife.it
Seraceni
S
S
Contini
C
C
D017088:AIDS-Related Opportunistic Infections / Q000134:cerebrospinal fluid / Q000469:parasitology; D000163:Acquired Immunodeficiency Syndrome / Q000382:microbiology; D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis / Q000134:cerebrospinal fluid / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002555:Cerebrospinal Fluid / Q000469:parasitology; D045647:DNA Glycosylases; D015342:DNA Probes; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics; D015800:Protozoan Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000469:parasitology; D016781:Toxoplasmosis, Cerebral / Q000134:cerebrospinal fluid / Q000469:parasitology
D000953:Antigens, Protozoan; D015342:DNA Probes; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C105531:SAG4 protein, Toxoplasma gondii; D029701:Saccharomyces cerevisiae Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C445668:MAG1 protein, S cerevisiae