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.
Subject(s)
DNA, Protozoan/blood , Fluorescence Resonance Energy Transfer/methods , Polymerase Chain Reaction/methods , Protozoan Proteins/genetics , Toxoplasma/isolation & purification , Toxoplasmosis/diagnosis , Animals , DNA Probes , DNA, Protozoan/analysis , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Sensitivity and Specificity , Taq Polymerase , Toxoplasma/genetics , Toxoplasma/immunology , Toxoplasmosis/parasitologyABSTRACT
A toxoplasmose infecta milhões de pessoas no mundo inteiro, sendo que a prevalência da infeccão humana na maioria dos países está entre 40 por cento e 50 por cento. No Brasil essa taxa aumenta até 80 por cento, dependendo da área estudada. Na maioria dos hospedeiros a infeccão é assintomática. A mulher grávida com sorologia negativa pode contribuir para o incremento da morbidade, transmitindo o Toxoplasma gondii para o feto, através da placenta, se adquirir toxoplasmose aguda durante a gravidez. O diagnóstico da toxoplasmose aguda é baseado na deteccão de IgM anti-Toxoplasma gondii circulante. A alta sensibilidade das técnicas sorológicas atuais trouxe a realidade da presenca de IgM residuais confundindo muitas vezes o diagnóstico final. Nesse sentido, as técnicas moleculares, tais como a reacão em cadeia da polimerase (PCR), podem ajudar a uma melhor interpretacão do estado real da interacão parasito/homem, embora sejam ainda pouco validadas para uso na rotina de diagnóstico laboratorial da toxoplasmose.
Subject(s)
Humans , Immunocompromised Host , Polymerase Chain Reaction , Serologic Tests , Toxoplasmosis, Congenital/diagnosis , Toxoplasmosis/diagnosis , Sensitivity and SpecificityABSTRACT
The polymerase chain reaction amplification of a fragment of the B1 gene of Toxoplasma gondii coupled to hybridization was performed in 42 patients from Rio de Janeiro, Brazil. The results showed 50% of positivity in the IgM positive toxoplasmosis group, and 12.5% in the positive IgG and negative IgM individuals. The data presented here revealed a lack of specificity of the molecular approach, clearly indicating that the primers used may co-amplify human sequences.