Soil is considered the primary source of Toxocara transmission to humans, especially children [14]. Soil contamination by eggs of Toxocara spp. is due to the presence of dogs and cats, two companion animals which are widely distributed in the world. This wide distribution of hosts is compounded by the features of Toxocara eggs, which can survive in the environment for months or even years [15]. It is therefore crucial to establish environmental surveillance programs aimed at detecting Toxocara eggs in soil, especially in places often crossed by dogs and cats and which happen to be frequented by children. However, detection of Toxocara eggs in soil samples still faces serious hurdles, which are in most parts due to the lack of a reliable sensitive method aimed at this objective. So far, enrichment by flotation and subsequent light microscopic examination remains the most used method for surveillance of soil contamination with Toxocara eggs. Needless to recall, this method is difficult to perform in the field, is time consuming and requires experience for light microscopy and parasitological diagnostics.
By using a DNA extraction method including a bead-beating step for disruption of the thick wall of Toxocara eggs and a clean-up step for removal of PCR inhibitors, we have managed to achieve a sensitivity of 6 eggs in 10 g of soil. To the best of our knowledge, this is an improvement compared to previously published methods [6, 9].
A method combining flotation and qPCR achieved a detection threshold of 10 eggs for the flatworm Echinococcus multilocularis per 10 g of soil but failed to reach a similar figure for Toxocara eggs [9]. Whereas the causes behind this low sensitivity for detection of Toxocara are not known, they might be associated with the use of methods which did not put emphasis on the mandatory disruption of Toxocara eggs prior to DNA extraction. By improving this step, we have been able to achieve a sensitivity level of Toxocara comparable to that observed with other roundworms. However, as shown by the results of spiking of soil, the total removal of PCR inhibitors cannot be guaranteed, as pointed out by other authors [16].
Another hurdle in the detection of control of soil contamination with Toxocara eggs is the non-homogenous distribution of Toxocara eggs in soil samples, even those sampled from the same backyards or playgrounds. As an illustration, samples 7 and 14 which came from the same playground did not display similar qPCR results. A solution might reside in sampling more areas. Also, PCR inhibitors as the cause of a PCR negative result cannot totally be ruled out. The same observation applies to samples 31, 32 and 40 which came from the same backyard. Of note, the alleged improved sensitivity on detection of helminth such as Echinococcus multilocularis was achieved by combining an enrichment (flotation) step with qPCR [9]. This renders the method cumbersome and time-consuming, two features which preclude the use of the method in the field for screening of hundreds of environmental samples.
There are several clean-up kits for removal of PCR inhibitors. In our assay, The AMPure® beads purification was compared with the PowerClean Pro Cleanup Kit (Qiagen, Hilden, Germany) and displayed similar efficiency with respect to removal of PCR inhibitors (data not shown). The AMPure® clean-up was favoured as it is compatible with the prospect of automation of the whole analytical processing. One lingering question is whether positive results from qPCR systematically can be taken at value face as a proof of soil contamination, given that dead eggs of Toxocara can be associated with positive qPCR signals without also being able to cause toxocarosis when ingested by humans. Whereas no definitive answer for this question is available, the fact that Toxocara eggs can survive in the environment for several years [15] and the probability that viable eggs can be mistaken for dead eggs upon light microscopic observation should prompt us to consider qPCR positive as a reliable marker of soil contamination with Toxocara eggs.