Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

Upper reference limits of high-sensitivity cardiac troponin T in a general population: comparison with those of sensitive cardiac troponin I.

BACKGROUND: Upper reference limits (97.5th, 99th percentiles) of high-sensitivity and sensitive cardiac troponins (hs-cTn and s-cTn) can be influenced by several factors. Our aim was to study: (1) the ability of hs-cTnT and s-cTnI to detect circulating cTn in a general community population, and (2) the effects of age, renal function, and gender on their 97.5th - 99th percentile values. METHODS: Hs-cTnT and s-cTnI values were measured in 177 subjects. RESULTS: Thirty-six subjects (20%) presented hs-cTnT values above the limit of detection (LoD), whereas no subjects presented detectable s-cTnI values. Men presented more frequently than women with detectable hs-cTnT levels (37% vs. 11%; p = 0.0001). Hs-cTnT was more frequently found in older (> or = 70 years) than in younger subjects (57 vs. 14%; p < 0.0001). Subjects with low estimated glomerular filtration rates (eGFR < 60 mL/min1/ 1.73m2) presented more frequently with detectable hs-cTnT levels than subjects with higher eGFR (71% vs. 17%; p < 0.0001). Hs-cTnT 97.5th - 99th percentiles varied according to selection by age, renal function, and gender; percentile values of s-cTnI were below the LoD of the assay. CONCLUSIONS: Hs-cTnT is more often quantified than s-cTnI in healthy subjects. Age, gender, and eGFR values influence 97.5th - 99th hs-cTnT percentile values.

Routine laboratory test enabling the detection of dermatophytes and the identification of Trichophyton rubrum by means of in-house duplex real-time PCR.

Dermatophytes are responsible, in majority, for fungal infections of skin, hair and nails, and Trichophyton rubrum is the most frequently isolated dermatophyte in humans. The time for dermatophyte growth in culture requires a total of two to four weeks. Molecular methods were developed to improve time to diagnosis and initiation of treatment. We present here an in-house duplex real-time PCR enabling detection of dermatophytes and simultaneous identification of T. rubrum from mycological samples and cultures. The objective of this work was to optimize the fungal DNA extraction method, the detection of dermatophytes and the identification of T. rubrum on a CFX96® (Real-Time PCR Detection System). In addition, the method comparison showed that this new method is more sensitive than the culture and microscopic observations. To conclude, this routinely used method has been accredited ISO 15189 since January 2020 in our laboratory.

Late diagnosis of congenital toxoplasmosis based on serological follow-up: A case report.

Toxoplasma gondii is a protozoan parasite infecting up to one third of the world's population. T. gondii infection is usually benign in immunocompetent patients but can be life-threatening when congenitally transmitted. Congenital toxoplasmosis presentation ranges from severe central nervous system and ocular features, to a well appearing newborn with onset of complications late in childhood. The diagnosis of subclinical form remains important since early treatment reduces later complications such as chorioretinitis. We report an atypical case of congenital toxoplasmosis with a delayed diagnosis, based on Toxoplasma-specific serological follow-up. The infant was born to a mother who became infected during pregnancy, thus inducing infant biological and clinical follow-up. Neither biological nor clinical arguments favored a diagnosis of congenital toxoplasmosis until ten months of life. Congenital toxoplasmosis was then suspected because of an unusual increase of specific IgG levels. Diagnosis was confirmed by detection of newly synthesized newborn Ig isotypes using complementary comparative mother-to-child immunological profile techniques and specific treatment therefore administered. This report highlights the importance to follow up newborns at risk of congenital toxoplasmosis with specific and newborn-appropriate techniques until Toxoplasma-IgG titers are completely negative. This allows not only the exclusion of congenital toxoplasmosis when serology becomes negative, but also the diagnosis and treatment of congenital toxoplasmosis when infection is detected later in development.