An improved test system for PCR-based specific detection ofEchinococcus multiloculariseggs

For the sensitive detection of eggs ofEchinococcus multilocularisin fox faeces by PCR we have evaluated a method based on the previous concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution. The eggs were microscopically detected in the fractions retained in 40 and 20µm mesh sieves. DNA of the taeniid eggs retained in the 20 µm sieve was obtained after alkaline lysis and PCR was performed usingE. multilocularisspecies-specific primers. Compared to the parasitological findings after examination of the small intestines of the foxes, the specificity of the PCR was 100% (no false-positive result with 20 foxes free ofE. multilocularis) and the sensitivity was 94% (33 positive results from total 35 foxes proven to be infected withE. multilocularis). Both false-negative results were obtained with faeces from foxes harbouring immature worms. Using faecal volumes between 2 and 20 ml, no inhibition of PCR was observed as was demonstrated by the amplification of size-modified target in parallel reactions. The tests were undertaken with fresh faeces stored in 70% ethanol, but egg detection by PCR was also possible after inactivation of eggs by freezing the faeces at −80°C for one week or by incubation at +70°C for 2 h.