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.
Centre National de Référence Toxoplasmose-Pôle Biologie Moléculaire, France.
PLoS One. 2021 Feb 17;16(2):e0246802. doi: 10.1371/journal.pone.0246802. eCollection 2021.
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.
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.
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.
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.
弓形虫-PCR 对于诊断眼部、脑部、播散性和先天性弓形虫病至关重要。本多中心研究评估了样本在 +4°C 下储存时间对 PCR 检测性能的影响，以便为样本在运输过程中或/和 PCR 前的储存提出建议。
五种基质，羊膜（AF）、脑脊液（CSF）和支气管肺泡灌洗液（BALF）、全血（WB）和血球（BC），被人工混入不同数量的刚地弓形虫（每毫升样本 20、100、500 个速殖子）或先前感染的 THP1 细胞。在 0 天和储存+4°C 后 2、4 和 7 天进行 DNA 提取。每个提取物至少用实时 PCR 扩增两次。
共研究了 252 个加标样本。在 4°C 下储存 7 天内，未观察到交叉点增加，所有样本在 AF、BALF、BC 和感染的 THP1 加标 WB 中均为阳性。对于 20 个寄生虫/毫升的 CSF 加标样本，只有 50%的 PCR 反应在 D7 时呈阳性（p<0.05）。对于 II 型寄生虫加标 WB，所有反应在 D7 时仍为阳性，但从 D2 起扩增明显延迟；对于 RH 株加标 WB，阳性反应的比例在 D7 时下降。
临床样本在+4°C 下储存与刚地弓形虫寄生虫的分子检测兼容。只要进行了两次 PCR 检测，样本可以储存长达 7 天。然而，从第五天开始，对于容易含有低寄生虫负荷的样本，我们建议进行多次 PCR 检测。
