A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

Abstract

bThis study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (C T ) obtained with the B1 qPCR was significantly higher (؉4.71 cycles) than that obtained with REP-529 qPCR (P < 0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean Toxoplasma quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay.T oxoplasmosis is a worldwide parasitic infection due to the intracellular parasite Toxoplasma gondii. The infection is usually asymptomatic in immunocompetent patients and more rarely results in fever, lymphadenopathy, or retinochoroiditis. In contrast, immunocompromised patients can experience severe neurologic, ocular, pulmonary, or disseminated disease (1). Yet, toxoplasmosis is well known for its pathogenicity during pregnancy. Indeed, when primary infection occurs in pregnant women, it can lead to congenital toxoplasmosis, with a frequency of transmission and a severity of fetal infection depending on the stage of pregnancy at which infection occurs (2). The diagnosis of toxoplasmosis is routinely based on serology. In some countries, such as France, seronegative pregnant women are monitored monthly by serology. In case of seroconversion, the detection of T. gondii DNA by PCR is a major diagnostic method for congenital toxoplasmosis and is performed on amniotic fluid (prenatal diagnosis) (3-5) and placenta or cord blood samples at birth (postnatal diagnosis) (6-8). In immunocompromised patients, DNA can also be found in cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL) fluid, or other samples, as guided by clinical signs. The 35-fold repeated B1 gene (9) has commonly been used for this molecular diagnosis since 1989, with acceptable sensitivity (3, 5, 10), but another sequence (REP-529, GenBank accession no. AF146527) was described more recently as being repeated 200 ...

What is your diagnosis? Corneal scrape from a dog

“…Serology for T gondii (IgG/IgM IFA; TVMDL, College Station, TX, USA) and Neospora caninum (IFA; TVMDL) were negative. PCR of the small corneal scrape sample, using primers specific for T gondii REP‐529 locus, yielded a faint band; however, DNA fragment extraction for sequencing to definitively confirm the protozoan was unsuccessful. The patient responded to topical antibiotics (ofloxacin, cefazolin), antiseptics (1% povidone‐iodine), and serum, in addition to oral anti‐protozoal therapy (ponazuril), which was elected due to extensive corneal vascularization.…”

Toxoplasmose ocular com reação em cadeia da polimerase positiva em sangue periférico – relato de dois casos, estado de São Paulo, Brasil

“…Reports on the importance of PCR to demonstrate the specificity and sensitivity necessary for the detection of genetic material of human microorganisms in biological samples have been published elsewhere [22,23,25,26,29,31,32,34,35]. However, this assay is still not requested in the ophthalmologic routine.…”

Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens

“…[13][14][15][16] Based on the premise that the sensitivity of qPCR is enhanced by the number of target sequences, the B1 gene (35 copies) 17 and the 529-bp repeat element (REP 529, more than 300 copies) 18 have been extensively used as targets for PCR detection. 16,19,20 Nevertheless, collection of some specimens, such as brain tissue or cerebrospinal fluid, represent a risk and are not always easily obtained. In this way, blood offers many advantages as a lowinvasive specimen and its usefulness in the diagnosis of toxoplasmosis by qPCR have been widely reported.…”