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

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 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.

Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples

“…Furthermore, in our laboratory, peripheral blood of patients suspected of acute toxoplasmosis was also analyzed ( (Guy & Joynson, 1995). In a study involving patients with acute toxoplasmosis in southeastern Brazil, the rate of parasite DNA-positive peripheral blood samples was 48.6% (17/35) (Kompalic-Cristo et al, 2007). Interestingly, both of these studies used B1 as the target gene, which is considered to be of lower sensitivity than the AF146527 marker that we used; however, the total number of analyzed samples was smaller and the patient selection criteria may have differed.…”

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

“…The inclusion in PCR assays of genes that constitute cells such as β-actin, β-globin, albumin and G3PD has been useful to evaluate the quality of samples in qPCR and PCR experiments (20,34,47,48). Due to the recognized need for evaluating the integrity of DNA, controls are usually used in separate reactions, generating more costs and consuming more time to establish the diagnosis (49).…”

Determining UV Inactivation of Toxoplasma gondii Oocysts by Using Cell Culture and a Mouse Bioassay

“…This study determines the mouse infectivity status primarily by animal health followed by a novel confirmation by a duplex qPCR assay instead of serology or immunohistochemistry. The B1 qPCR assay used to detect T. gondii in infected tissues was adopted from an assay developed by Kompalic-Cristo et al (39). This approach is very reliable and less labor-intensive than serology and immunohistochemistry and resulted in a dual detection of both mouse tissue and the presence or absence of T. gondii.…”