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Parasitology Research

, Volume 101, Issue 3, pp 619–625 | Cite as

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

  • Alicia Kompalic-Cristo
  • Cassia Frotta
  • Martha Suárez-Mutis
  • Octavio Fernandes
  • Constança BrittoEmail author
Original Paper

Abstract

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.

Keywords

Toxoplasmosis Parasite Burden Polymerase Chain Reaction Result Conventional Polymerase Chain Reaction Positive Polymerase Chain Reaction Result 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgement

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.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Alicia Kompalic-Cristo
    • 1
    • 2
  • Cassia Frotta
    • 3
  • Martha Suárez-Mutis
    • 1
  • Octavio Fernandes
    • 1
    • 3
  • Constança Britto
    • 4
    Email author
  1. 1.Laboratório de Epidemiologia Molecular de Doenças Infecciosas, Departamento de Medicina TropicalInstituto Oswaldo CruzRio de JaneiroBrazil
  2. 2.Departamento de Medicina Preventiva y SocialUniversidad Centroccidental Lisandro Alvarado, Decanato de MedicinaBarquisimetoVenezuela
  3. 3.Núcleo Técnico OperacionalDiagnósticos da AméricaRio de JaneiroBrazil
  4. 4.Laboratório de Biologia Molecular e Doenças Endêmicas, Departamento de Bioquímica e Biologia MolecularInstituto Oswaldo CruzRio de JaneiroBrazil

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