Egg intensity and freeze-thawing of fecal samples affect sensitivity of Echinococcus multilocularis detection by PCR

Echinococcus multilocularis is one of the most relevant zoonotic parasites with about 18,000 human cases per year. Its detection in wild host is crucial for disease prevention. The present study aimed to determine factors affecting the sensitivity of E. multilocularis detection by PCR using DNA extracted from fecal samples of coyotes (Canis latrans). Fecal samples were screened for the presence of Taeniidae eggs through centrifugation and sedimentation. DNA was extracted from fecal samples with and without prior freeze-thawing of the sample and then subjected to PCR targeting a mitochondrial gene (nad1) and a multi-loci microsatellite marker (EmsB). The presence of PCR inhibitors was determined through internal amplification control. Subjecting the sample to repeated freeze-thaw cycles significantly increased the sensitivity of the PCR by 20%. Likewise, egg intensity had a significant effect on PCR, an effect which was more pronounced for samples not subjected to freeze-thawing. Two or more eggs per gram of feces significantly increased the odds for a positive PCR outcome. The presence of PCR inhibitors had no effect on PCR in samples subjected to freeze-thaw cycles, whereas in samples not subjected to freeze-thaw cycles, the presence of PCR inhibitors was associated with a 0.78 lower odds ratio of positive PCR outcome. Targeting a nuclear versus a mitochondrial gene did not have a significant effect on the sensitivity of PCR. We recommend freeze-thawing samples prior to DNA extraction to become a standard procedure for E. multilocularis detection in canid feces.