Deliah Tamsyn Winterfeld (Greifswald / DE), Majda Globokar (Kornwestheim / DE), Nikola Pantchev (Kornwestheim / DE), Birgit Schauer (Greifswald / DE), Susan Mouchantat (Greifswald / DE), Prof. Dr. Franz J. Conraths (Greifswald / DE), Jana Schulz (Greifswald / DE), Dr. Gereon Schares (Greifswald / DE), Dr. med. vet. Pavlo Maksimov (Greifswald / DE)
Abstract text
Introduction:
Toxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. Embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children. Little is known about the diagnostic performance and comparison of different detection methods of Toxocara spp. in faeces of cats and dogs.
Objectives:
The aim of this study is to assess the analytical sensitivity of different methods for the detection of Toxocara spp. in faeces of cats and dogs. In addition, a high-throughput method has been developed to facilitate epidemiological studies at the species level.
Materials & methods:
Two automated DNA extraction methods using mechanical lysis and the King Fisher® flex system were compared regarding their performances. DNA was assessed for parasite-specific genome sequences by TaqMan® real-time PCR. All faecal samples were also examined using the sedimentation-flotation method to allow comparisons between molecular and conventional parasitological methods. Furthermore, a sieving method for the detection, enrichment and purification of parasite eggs in faecal samples was developed and subsequently applied.
Results:
The analytical sensitivity of real-time PCR in combination with automated DNA extraction methods was higher if King Fisher® in 96-well-plates was applied (limit of detection: < 1 egg/extraction [200 µl] (T. cati), 3 eggs/extraction [200 µl] (T. canis)) as compared to the use of 24-deep-well-plates (limit of detection: 2 eggs/extraction [1 ml] (T. cati), 28 eggs/extraction [1 ml] (T. canis)). The frequently used sedimentation-flotation method had a slightly lower analytical sensitivity (limit of detection: 78 eggs/sample) compared to the new established sieving method (limit of detection: 10 eggs/sample).
Conclusion:
Real-time PCR in combination with the automated DNA extraction in 96-well-plates and the sieving method had comparable analytical sensitivities. The sieving method was developed to purify and enrich parasite eggs, with a higher detection sensitivity than the sedimentation-flotation method. However, the method based on the automated DNA extraction and subsequent real-time PCR provides a significantly higher sample throughput. Thus, it allows the examination of at least five times more samples at the same time at species level. Therefore, this method is markedly more advantageous than the conventional flotation method from a labour, economic and epidemiological point of view.