title: Evaluation of a new 5'-nuclease real-time PCR assay targeting the Toxoplasma gondii AF146527 genomic repeat.
abstract: Toxoplasma gondii can be responsible for congenital toxoplasmosis leading to mild or severe sequelae, and for life-threatening infections in immunocompromised hosts. A new 5'-nuclease real-time PCR assay that targets the 300-fold repeated AF146527 DNA sequence (TaqMan-AF-PCR) has been developed and its performance for diagnosis of toxoplasmosis and treatment follow-up has been assessed. A retrospective analysis was first performed with 144 clinical specimens previously analysed for the presence of T. gondii DNA by a PCR-ELISA assay that targets the B1 gene of T. gondii (B1-PCR-ELISA). Fifteen samples, all from patients with clinically proven toxoplasmosis, were negative according to B1-PCR-ELISA and positive according to TaqMan-AF-PCR. A prospective analysis was then performed with 203 consecutive clinical specimens received at the laboratory of Parasitology of Saint-Louis Hospital during a 4-month period. The diagnosis of toxoplasmosis in two patients was made according to the TaqMan-AF-PCR whereas the B1-PCR-ELISA failed to make diagnosis. Additionally, iterative samples from a patient with cerebral and disseminated toxoplasmosis, already tested using a B1 real-time PCR assay, were tested using the TaqMan-AF-PCR and a Light Cycler real-time PCR assay targeting the same repetitive AF146527 sequence (LC-AF-PCR). Detection was achieved with the TaqMan-AF-PCR, with a mean gain of 7.1 and 3.3 amplification cycles when compared with the B1 real-time PCR and the LC-AF-PCR, respectively. This study demonstrates the higher sensitivity of the 5'-nuclease real-time PCR assay developed for the AF146527 DNA sequence and confirms the interest of using this highly repeated target to improve the diagnosis of toxoplasmosis.
authors.affiliation: Laboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris and Université Paris-Diderot, France. jean.menotti@sls.ap-hop-paris.fr
authors.forename: J
authors.initials: J
authors.lastname: Menotti
authors.forename: Y J-F
authors.initials: YJ
authors.lastname: Garin
authors.forename: P
authors.initials: P
authors.lastname: Thulliez
authors.forename: M-C
authors.initials: MC
authors.lastname: Sérugue
authors.forename: J
authors.initials: J
authors.lastname: Stanislawiak
authors.forename: P
authors.initials: P
authors.lastname: Ribaud
authors.forename: N
authors.initials: N
authors.lastname: de Castro
authors.forename: S
authors.initials: S
authors.lastname: Houzé
authors.forename: F
authors.initials: F
authors.lastname: Derouin
mesh_terms: D001483:Base Sequence; D006801:Humans; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods*; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis*