Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in human peripheral blood
- 698 Downloads
In this paper, we examined the diagnostic value of a real-time polymerase chain reaction (PCR) using fluorescence resonance energy transfer (TaqMan assay) with a new set of primers and probe targeting the B1 gene to reproducibly detect and quantify Toxoplasma gondii in human blood. A total of 183 buffy coat samples from patients serologically classified as recent toxoplasmosis (immunoglobulin M (IgM)+, n = 35) or chronic infection (IgM− and immunoglobulin G (IgG)+, n = 110), and seronegative individuals (n = 38) was investigated. Of the IgM seropositive patients, 17:35 (48.6%) presented parasitaemia, whereas 3.6% positivity was achieved in those individuals that theoretically corresponded to chronic infection (4:110). In the seronegative group, the assay provided 7.9% (3/38) of positive results. Interestingly, one of them was confirmed as positive in a conventional PCR targeting the Toxoplasma B1 gene after hybridization with an internal probe. Real-time PCR was able to accurately quantify the parasite load when concentrations of T. gondii DNA are low, revealing a parasite burden ranged from 9.92 × 10−3 to 8.73 × 10−1 tachyzoites genome per milliliter of blood. The chance of an IgM+ patient to present parasitemia detected by the TaqMan procedure was 19.02 times greater than in IgM− individuals (P < 0.05). It was observed a positive association between the optical density values of the IgM serological tests and the number of circulating parasites in the acute patients (P < 0.0001). The specificity of the molecular test was 95.3% when calculated using IgM+ patients as disease group and IgM− as nondisease group. The low sensitivity observed in the IgM seropositive group (48.6%) could be due to the use of buffy coat as clinical material for DNA extraction. An amplification control based on the human β-actin gene was used in parallel to monitor PCR inhibition and to control for DNA integrity.
KeywordsToxoplasmosis Parasite Burden Polymerase Chain Reaction Result Conventional Polymerase Chain Reaction Positive Polymerase Chain Reaction Result
This study was partially supported by a grant from the SUS/CNPq/FAPERJ program. Alicia Kompalic-Cristo is a CONICIT and UCLA fellow, and Constança Britto is a CNPq fellow. We thank the PDTIS/FIOCRUZ framework for the PCR real-time workstation facilities, Ms. Maria Angelica Cardoso for technical assistance, and Dr. Claudia M. d’Avila-Levy for carefully reading this manuscript. The experiments performed herein comply with the current laws of Brazil.
- Contini C, Cultrera R, Seraceni S, Segala D, Romani R, Fainardi E, Cinque P, Lazzarin A, Delia S (2002) The role of stage-specific oligonucleotide primers in providing effective laboratory support for the molecular diagnosis of reactivates Toxoplasma gondii encephalitis in patients with AIDS. J Med Microbiol 51:879–890PubMedGoogle Scholar
- Costa JM, Pautas C, Ernault P, Foulet F, Cordonnier C, Bretagne S (2000) Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. J Clin Microbiol 38:2929–2932PubMedGoogle Scholar
- Martino R, Bretagne S, Einsele H, Maertens J, Ullmann AJ, Parody R, Schumacher U, Pautas C, Theunissen K, Schindel C, Munoz C, Margali N, Cordonnier C (2005) Early detection of Toxoplasma infection by molecular monitoring of Toxoplasma gondii in peripheral blood samples after allogeneic stem cell transplantation. Clin Infect Dis 40:67–78PubMedCrossRefGoogle Scholar
- Mei-Hui L, Tse-Ching C, Tseng-Tong K, Ching-Chung T, Ching-Ping T (2000) Real-Time PCR for quantitative detection of Toxoplasma gondii. J Clin Microbiol 38:4121–4125Google Scholar
- O’Driscoll JC, Holliman RE (1991) Toxoplasmosis and bone marrow transplantation. Rev Med Microbiol 2:215–222Google Scholar
- Pelloux H, Dupoy-Camet J, Derouin F, Aboulker JP, Raffi F, the Bio-Toxo Study Group (1997) A multicentre prospective study for the polymerase chain reaction detection of Toxoplasma gondii DNA in blood samples from 186 AIDS patients with suspected toxoplasmic encephalitis. AIDS 11:1888–1890PubMedCrossRefGoogle Scholar
- Romand S, Chosson M, Franck J, Wallon M, Kieffer F, Kaiser K, Dumon H, Peyron F, Thulliez P, Picot S (2004) Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii. Am J Obstet Gynecol 190:797–802PubMedCrossRefGoogle Scholar