| Abstract: | Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.Molecular methods play an important role in the microbiological diagnosis of infectious diseases due to their high sensitivity and specificity (1, 5). Among these, PCR is now recognized as an essential diagnostic tool for congenital toxoplasmosis (1, 8, 11, 19, 20) as well as for toxoplasmosis in immunocompromised individuals (9, 16) and ocular toxoplasmosis (22, 26). Multicenter comparative evaluations have shown sensitivity differences in the PCR detection of Toxoplasma gondii in amniotic fluid (AF), especially when parasite loads were low (<10 T. gondii cells/ml [T/ml]) (2, 14). Since it has been reported that about half of AF samples infected by T. gondii contain fewer than 10 T/ml (8), it is important to use procedures that allow for the detection of such parasite concentrations.Among the different steps that participate in ensuring a reliable molecular detection method, DNA extraction is crucial (3, 12, 24, 27). Indeed, prior to amplification, biological samples (tissue biopsy samples, body fluid samples, tissue scrapings, etc.) must be prepared not only to extract and concentrate the DNA but also to eliminate proteins, lipids, polysaccharides, and other potential inhibitors of the DNA polymerase. DNA extraction consists of nucleic acid isolation, purification, and concentration in an eluted product, and many commercial systems have now become available, replacing in-house methods in many laboratories (25).One of the general aims of the French National Reference Centre for Toxoplasmosis (Centre National de Référence de la Toxoplasmose) is to determine the best molecular detection strategies for Toxoplasma and to recommend them to diagnosis laboratories. In this sense, a specific objective is to better define the importance of DNA extraction in the whole PCR process. This prompted us to compare the performance of widely used commercial DNA extraction kits for T. gondii detection using a strict experimental protocol. We carried out a prospective and multicenter study to evaluate five commercial DNA extraction procedures, two manual and three automated kits, for the isolation of T. gondii DNA in AF. The yield of DNA extraction was assessed by subsequent DNA amplification, combining two Toxoplasma-specific real-time PCR assays using either TaqMan or fluorescence resonance energy transfer (FRET) probe detection, as well as one human beta-globin PCR and one inhibitor search PCR. To detect fine differences among the extraction methods, we decided to work with low concentrations of the parasite, down to 5 tachyzoites/ml (2, 14). |
