Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in human peripheral blood.
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 x 10(-3) to 8.73 x 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 beta-actin gene was used in parallel to monitor PCR inhibition and to control for DNA integrity.
Kompalic-Cristo
Alicia
A
Laboratório de Epidemiologia Molecular de Doenças Infecciosas, Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil.
Frotta
Cassia
C
Suárez-Mutis
Martha
M
Fernandes
Octavio
O
Britto
Constança
C
D000818:Animals; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis / Q000097:blood*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis* / Q000469:parasitology
D015342:DNA Probes; D016054:DNA, Protozoan; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D015800:Protozoan Proteins; D019914:Taq Polymerase