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Detection and genotyping of Toxoplasma gondii by real-time PCR and pyrosequencing

Abstract number: s16

Edvinsson  B., Lappalainen  M., Evengård  B., for the European Study Group on Toxoplasmosis 

Objectives: 

Toxoplasma gondii is a protozoan parasite with worldwide distribution. Primary infections in humans are usually asymptomatic or characterized by non-specific symptoms. The infection is, however, severe or fatal in patients with a suppressed immune system, and is here most often caused by chronic infections that become reactivated. Rapid and sensitive diagnosis by PCR may be of importance for the survival of these patients. PCR techniques used to detect the parasite in patient samples are not yet standardized. We have compared the performance of two real-time PCR assays targeting two different repetitive DNA elements of T. gondii. We also aimed to develop a pyrosequencing assay to discriminate between the three main genotypes of T. gondii; i.e. type I, II and III.

Methods: 

The sensitivities for detecting T. gondii by real-time PCR when targeting the 35-fold repeated B1 gene or a 200–300 fold repeated 529 bp elements were compared. The impact on the diagnostic sensitivity when using two different types of internal amplification controls, either amplified by the primers used for detection of T. gondii or by a different set of primers, was tested. For genotyping, real-time PCR and pyrosequencing were used to analyse a fragment of the GRA6 gene in 21 T. gondii isolates.

Results: 

The sensitivity was higher when the 200–300 fold repeated 529 bp element was used. The diagnostic performance of the real-time PCR targeting the 529 bp elements was not affected by inclusion of an internal amplification control. The Pyrosequencing assay discriminated between type I, II and III T. gondii.

Conclusions: 

Real-time PCR, including an internal amplification control, can be used to detect low amounts of T. gondii in a sample by amplification of a part of the 529 bp elements. This may be of importance if the aim is a sensitive and rapid diagnosis of toxoplasmosis in immunocompromised patients. Pyrosequencing can be used for rapid and high throughput genotyping of the parasite in clinical findings, and should help to further elucidate if there are differences in the clinical manifestations of toxoplasmosis caused by type I, II or III T. gondii.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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