Toxocarosis is a zoonotic disease caused by the larvae of Toxocara, a worldwide distributed roundworm genus of the ascaroid group. Toxocara species of human and animal health significance are essentially represented by T. canis and T. cati, parasites of canids and felids, respectively. According to recent data, the prevalence of T. canis or T. cati is variable but remains high. This widespread prevalence of Toxocara spp. in dogs and cats is associated with the contamination of playgrounds, municipal parks and households with eggs[3–5]. Red foxes (Vulpes vulpes) are also frequently infected by T. canis, an observation to consider in the light of recent epidemiological studies, which point out the progressive increase in the number of foxes in European urban environments over the last few years[6, 7]. Children are most likely infected through ingestion of embryonated eggs of T. canis or T. cati when playing on soils contaminated with dogs or cats feces containing Toxocara eggs. After ingestion, Toxocara eggs hatch in intestine and release larvae (juvenile worms) that penetrate the small intestine wall to enter the bloodstream. They subsequently travel through the bloodstream to all the major organs. Although most infections are asymptomatic, two well-defined syndromes are classically recognized in humans: visceral larva migrans (VLM), a systemic disease caused by larval migration through major organs, and ocular larva migrans (OLM), a disease limited to the eye and optic nerve. Less severe syndromes have been described, in children (covert toxocariasis) and in adults (common toxocariasis)[8–10].
Accordingly, monitoring the presence of Toxocara eggs in dogs and cats feces, as well as in playgrounds and municipal parks likely to be contaminated by animal stools is critical in the control of toxocarosis.
Microscopic examination of dog and cat stools or soil samples is commonly used for identification of Toxocara eggs. The method includes an enrichment pre-analytical step through the use of centrifuge-flotation techniques. However, the method displays poor sensitivity, due to the low recovery of Toxocara eggs especially from soil samples. Furthermore, microscopic examination can fail to unambiguously discriminate eggs of Toxocara species because they are fairly similar[12, 13]. Other important roundworms include Baylisascaris procyonis, the common intestinal roundworm of raccoons responsible for a severe human neurologic disease and possibly Toxocara vitulorum (T. vitulorum), a cattle roundworm, which has been linked to a low level zoonosis alleged to affect children. There is, however, much uncertainty about the zoonotic potential of this species, as infections attributed to T. vitulorum could be due to T. canis or T. cati. Owing to their high sensitivity and high specificity, PCR methods have been highlighted to improve the detection and identification of Toxocara species of human significance. Numerous PCR methods for the detection and identification of T. canis and T. cati were reported to identify T. canis and T. cati in dog, fox and cat stools, as well as in soil samples[16, 17]. The DNA-based methods take advantage of the high genetic variability within molecular markers such as ITS2 for the discrimination of T. canis and T. cati from their closely-related neighbors, namely T. leonina, T. vitulorum and T. malaysiensis[2, 17]. However, despite these achievements, many drawbacks actually preclude their implementation in routine screening of Toxocara spp of clinical significance, either in dog, cat and fox stools or in soil samples from playgrounds and parks. These pitfalls include the risk of carry over contamination, the low throughput of samples analysis, the difficulty of automation and the lack of standardization. Accordingly, real-time PCR has the potential to circumvent the drawbacks of endpoint PCR. Moreover, real-time PCR is rapid and can allow analysis of many samples in a short time without any need of additional post-PCR manipulations, often responsible for carry-over contamination. Consequently, the development of a real-time PCR assay for detection of Toxocara eggs could improve diagnosis of toxocariosis and thus improve health status of children in contaminated areas.
In the current study, we have developed a 2qPCR assay for rapid and specific identification of T. canis and T. cati eggs in fecal samples as well as in soil samples from sandpits and playgrounds. Results suggest that the 2qPCR assay is sensitive and specific for detecting T. canis and/or T. cati both in fecal and soil samples.