Parasitology Freezing and storage at -20 °C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis Laurence Delhaes a, b , Denis Filisetti b, c , Marie-Pierre Brenier-Pinchart b, d , Hervé Pelloux b, d , Hélène Yéra b, e , Frédéric Dalle b, f , Yvon Sterkers b, g, h , Emmanuelle Varlet-Marie b, g , Feriel Touafek b, i , Sophie Cassaing b, j , Patrick Bastien b, g, h, ⁎ a CHU/Université de Lille 2, Département de Parasitologie-Mycologie, BDEEP-EA4547, Lille, France b Pôle “Biologie Moléculaire” du Centre National de Référence de la Toxoplasmose, Montpellier, France c Hôpitaux Universitaires de Strasbourg/Université de Strasbourg, Laboratoire de Parasitologie et Mycologie Médicale, Strasbourg, France d CHU de Grenoble/Université Joseph Fourier, Laboratoire de Parasitologie et Mycologie, Grenoble, France e Université Paris Descartes/Assistance Publique des Hôpitaux de Paris, Hôpital Cochin, Département de Parasitologie-Mycologie, Paris, France f CHU de Dijon/Université de Bourgogne, Département de Parasitologie-Mycologie, Dijon, France g CHRU/Université Montpellier 1, Département de Parasitologie-Mycologie, Montpellier, France h CNRS UMR5290/IRD 224/UM1/UM2 ("MiVEGEC"), Montpellier, France i Assistance Publique des Hôpitaux de Paris, Hôpital Pité Salpêtrière - Charles Foix, Laboratoire de Parasitologie et Mycologie, Paris, France j CHU de Toulouse, Département de Parasitologie-Mycologie, Toulouse, France abstract article info Article history: Received 4 April 2014 Received in revised form 6 July 2014 Accepted 21 August 2014 Available online 29 August 2014 Keywords: Real-Time PCR DNA storage Toxoplasma gondii Amniotic fluid Congenital toxoplasmosis Nucleic acid-based testing has become crucial for toxoplasmosis diagnosis. For retrospective (forensic or scientif- ic) studies, optimal methods must be employed for DNA long-term storage. We compared Toxoplasma gondii detection before and after DNA storage using real-time PCR. No significant differences were found depending on duration or storage conditions at -20 °C or -80 °C. © 2014 Elsevier Inc. All rights reserved. Molecular diagnosis (PCR) is now recognized as an essential tool for the diagnosis of congenital toxoplasmosis (Bastien, 2002). In this condi- tion, the search for Toxoplasma gondii DNA is done prenatally using amniotic fluid (AF) samples. The sensitivity of this molecular diagnosis is not 100%, as the best current PCR assays do not detect 10–20% of the congenital toxoplasmosis cases (Delhaes et al. 2013; Sterkers et al. 2012), and parasitic loads in AF may indeed be extremely low, with a median below 10 T. gondii cells per mL according to previous publica- tions (Bastien et al., 2007; Costa et al. 2001; Kaiser et al., 2007; Romand et al. 2004). DNA extraction from such samples having low parasitic loads may have a low yield (Yera et al., 2009), leading to difficulties in diagnosis that are enhanced if some DNA degradation occurs after extraction. Consequently, it is critical that optimal methods are employed either for short-term storage (if diagnostic analysis is delayed on the short-term) or long-term storage (compulsory conservation in France, retrospective analysis). For example, retrospective examination of a previously extracted DNA sample may be necessary for forensic rea- son or for further scientific studies. Here, we report a multicenter study that aimed at evaluating the effect of long-term conservation of Toxo- plasma DNA extracted from AF by comparing the parasite detection be- fore and after DNA storage using real-time PCR, in order to propose suitable and documented recommendations. A total of 53 PCR-positive AF sample DNAs were retrospectively recruited from documented congenital toxoplasmosis cases in 8 profi- cient centers that form part of the molecular biology ‘pole’ of the French National Reference Center for Toxoplasmosis (http://www.chu-reims. fr/professionnels/cnr-toxoplasmose-1). These centers assess molecular methods used for the diagnosis of congenital toxoplasmosis and propose technical recommendations to the medical and scientific community (Sterkers et al., 2010). The AF samples were isolated by amniocentesis performed at a mean of 18 ± 10 weeks of amenorrhea (WA). DNA extractions from AF were done according to manufacturers' recommendations, using either QIAmp DNA Mini Kit (Qiagen®, Courtaboeuf, France) in 6 centers, High Pure Template Preparation kit (Roche Diagnostics®, Meylan, France) in 1 center, or the Tween-Nonidet-NaOH (0.5% Tween 20, 0.5% Diagnostic Microbiology and Infectious Disease 80 (2014) 197–199 ⁎ Corresponding author. Tel.: +33-4-67-33-23-50; fax: +33-4-67-33-23-58. E-mail addresses: patrick.bastien@univ-montp1.fr, p-bastien@chu-montpellier.fr (P. Bastien). http://dx.doi.org/10.1016/j.diagmicrobio.2014.08.007 0732-8893/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio