Marie-Pierre Brenier-Pinchart1,2, Emmanuelle Varlet-Marie2,3, Florence Robert-Gangneux2,4, Denis Filisetti2,5, Juliette Guitard2,6, Yvon Sterkers2,7, Hélène Yera2,8, Hervé Pelloux1,2, Patrick Bastien2,7. 1. Laboratoire de Parasitologie-Mycologie, CHU Grenoble Alpes et Institut pour l'Avancée des Biosciences (IAB), INSERM U1209-CNRS UMR 5309, Université Grenoble Alpes Grenoble, Grenoble, France. 2. Centre National de Référence Toxoplasmose-Pôle Biologie Moléculaire, France. 3. Université de Montpellier et Laboratoire de Parasitologie-Mycologie CHU Montpellier, Montpellier, France. 4. CHU Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé Environnement Travail), UMR_S 1085, Université de Rennes, Rennes, France. 5. Institut de Parasitologie et de Pathologie Tropicale, Université de Strasbourg et Laboratoire de Parasitologie et Mycologie Médicale, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. 6. Inserm, Centre de Recherche Saint-Antoine, CRSA, AP-HP, Hôpital Saint-Antoine, Sorbonne Université, Paris, France. 7. CNRS, IRD, CHU de Montpellier, "MiVEGEC" et Laboratoire de Parasitologie-Mycologie, Université de Montpellier, Montpellier, France. 8. Laboratoire de Parasitologie-Mycologie, Hôpital Cochin, Université de Paris, AP-HP, Paris, France.
Abstract
INTRODUCTION: Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR. MATERIALS AND METHODS: Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infected THP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR. RESULTS: A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infected THP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7. CONCLUSION: The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.
INTRODUCTION:Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR. MATERIALS AND METHODS: Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infectedTHP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR. RESULTS: A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infectedTHP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7. CONCLUSION: The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.
Authors: C Dard; S Bailly; T Drouet; H Fricker-Hidalgo; M P Brenier-Pinchart; H Pelloux Journal: J Microbiol Methods Date: 2017-01-16 Impact factor: 2.363