Impact of pre-analytic step duration on molecular diagnosis of toxoplasmosis for five types of biological samples

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.

Fig 1. PCR performance scores observed for different samples and parasite concentrations (per mL).

A: AF, BAL and ’small WB + PRU’; B: CSF; C: ’large WB + RH’; D: ’large WB + infected-THP1’; E: BC. AF: amniotic fluid; BALF: bronchoalveolar lavage fluid; WB: whole blood; CSF: cerebrospinal fluid; BC: buffy coat; Tg: Toxoplasma gondii; RH: RH Toxoplasma strain; PRU: Prugnaud Toxoplasma strain. *p< 0.05 compared to PCR performance scores without conservation (D0).

Fig 2. Crossing point values of PCR performed on seven different T. gondii-spiked samples without and after storage at +4°C during 2, 4 and 7 days before processing for molecular diagnosis of toxoplasmosis.

A: BALF and AF; B: CSF; C: ’large WB + RH’ and ’large WB + infected-THP1’; D: BC and ’small WB + PRU’. For each storage duration at + 4°C and T. gondii concentration (Tg/mL), three different samples were extracted in parallel and at least 2 PCR were performed for each sample. Cp means ± SD were reported only if the PCR performance score was 100%. Tg: Toxoplasma gondii; Cp: crossing point; SD: standard deviation; AF: amniotic fluid; CSF: cerebrospinal fluid; BALF: bronchoalveolar lavage fluid; WB: whole blood; BC: buffy coat; RH: RH Toxoplasma strain. * p<0.05 compared with Cp measured in samples without conservation.