Optimization of Toxoplasma gondii DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit

Abstract

Antenatal diagnosis of congenital toxoplasmosis relies on PCR in amniotic fluid. Because parasitic load is often low, DNA extraction must be optimized. Manual methods remain widespread although automated methods appear more effective. This study aimed at optimizing an automated method and at comparing it with a widespread manual method: QIAamp DNA minikit. To optimize NucliSens easyMAG, we evaluated the addition of proteinase K pre-treatment and the increase of the amount of silica particles used for the extraction. The optimized method was then compared to QIAamp DNA minikit on samples containing less than 25 tachyzoites/ml. NucliSens easyMAG DNA yield was improved after proteinase K pre-treatment (p < 0.01), but not with a higher silica particle input. The optimized method yielded more positive PCRs than the manual method, especially for samples containing 5 tachyzoites/ml or less (71% vs 26%, p < 10(-4)). The DNA amount in samples found positive by PCR was higher after optimized automated extraction than after manual extraction (p < 10(-4)). Proteinase K pre-treatment should be added to extract DNA from amniotic fluid using NucliSens easyMAG. Using this optimized automated method rather than manual methods would improve the sensitivity of Toxoplasma PCR and simplify the daily workflow.

Diagnosing congenital toxoplasmosis: where are we? A systematic review

“…Table 1 provides an overview of all studies included in the final sample and of all data elements used during the data analysis process. The 41 studies were distributed into the previously determined three categories as follows: DIAGNOSTIC METHODS [2,4,5,8,9,12,14,16,17,20,25,27,29,32,34,35,37,41,42 (nineteen studies)]; DIAGNOSIS IN PREGNANT WOMEN [ 3,7,10,14,25,27,35,42,46,50,51,53, 59 thirteen studies)]; DIAGNOSIS IN NEONATES [7,8,11,20,61,64,65,67,68,70,71,72 (twelve studies)] and DIAGNOSIS IN INFANTS [1,3,5,9,11,12,20,27,…”

“…The extreme diversity of methods and performances of Toxoplasma PCR assays makes the use of commercial PCR kits an attractive alternative, as they offer a chance for standardization [2]. Toxoplasma PCR in amniotic fluid (AF) is the cornerstone of the antenatal diagnosis of congenital toxoplasmosis [36,37]. Highly sensitive methods are necessary because up to 46% of infected AF contain less than ten tachyzoites per milliliter (T/ml) [38,37].…”

Zebra mussel as a new tool to show evidence of freshwater contamination by waterborneToxoplasma gondii

“…DNA extraction was optimized between October and March, as suggested in Faucher et al . (). Using this last method, sample purification is more efficiently and final volume is reduced of about 10 fold which improve the sensitivity of Toxoplasma PCR.…”

“…Then the cells were lysed by boiling for 8 min at 99°C, and samples were centrifuged at 11 000 g for 3 min, and then the supernatants were stored at À80°C until detection by qPCR. DNA extraction was optimized between October and March, as suggested in Faucher et al (2012). Using this last method, sample purification is more efficiently and final volume is reduced of about 10 fold which improve the sensitivity of Toxoplasma PCR.…”

Comparison of Mother and Child Antibodies That Target High-Molecular-Mass Toxoplasma gondii Antigens by Immunoblotting Improves Neonatal Diagnosis of Congenital Toxoplasmosis

“…The samples of the 118 infected children were consecutively collected from 1999 to 2010 whereas the samples of the 118 noninfected children were drawn randomly from all cases with CT excluded in the same period. In 145 cases, amniotic fluid had been sampled after 16 weeks of gestation and at least 4 weeks after the estimated date of maternal infection for PCR targeting the 529-bp repeat element (6,21) and/or mouse inoculation. Comparison of mother and child profiles using cord blood (Marseille) or peripheral blood sampled at 3 days of life (Lyon) had been performed prospectively with the Toxoplasma WB IgG IgM assay (LD-Bio, Lyon, France) according to the manufacturer's recommendations.…”