ABC series on diagnostic parasitology part 1: the Willis method

Diagnosis of parasitic infections in animals is an exciting and stimulating challenge. There are several ways to diagnose parasitic infections: by direct identification of the parasite, by serology or using molecular methods.

To perform direct identification of parasites, particularly ova present in faeces, it is possible to use faecal smears, flotation and sedimentation tests. Morphology and dimensions are the major diagnostic criteria to which a microscope is key in the veterinary clinic. The faecal flotation of parasitic forms enables confirmation of these criteria and is therefore routinely used for the detection of most parasites residing within the gastrointestinal tract (Foreyt, 1989). Methods based on flotation are fast and inexpensive, and can be quickly implemented as a measure for infection control (Sousa et al, 2016). Despite these advantages, there are also some limitations in these methods:

The Willis method (Willis, 1921) is a flotation method, used for detection of light structures such as protozoan cysts and light helminth eggs (Carvalho et al, 2012; Sousa et al, 2016). This assay has been the most commonly used technique for detecting these parasitic structures (Cerqueira et al, 2007). It was nearly a century ago, in 1921 that H. Hastings Willis first published an improved method to reliably detect hookworm ova, providing a significantly higher percentage of positive results than the previously used smear and centrifuge methods (or their combination) (Willis, 1921). The Willis method, published in the reputable Medical Journal of Australia, was based on two wellknown assumptions: that the eggs float in a salt solution of approximately 1.130 specific gravity; and adhere to a glass surface with which they come into contact. This last property of egg adhesion was described in 1908 by William Pepper, an Assistant Professor of Clinical Pathology, University of Pennsylvania. Pepper had recently treated two cases of uncinariasis (Uncinaria duodenale and Uncinaria americana), and while making frequent examinations of the patient's stools, Pepper noted that the parasites' ova were quite sticky and that this peculiarity could be made use of in searching for them. In particular, Pepper proposed that a drop of the washed sedimented faeces be allowed to stand on a slide for a few minutes and then immersed in water and examined microscopically, after which only the eggs would be found adhering to the slide, and all other material would have washed away. Willis, taking into consideration Peppers findings, used a container measuring 3.3 cm in diameter and 0.8 cm in height; filled about one-sixth with stools to be examined, and the rest with salt solution to which stools were thoroughly mixed. The final step focused on filling the solution to the brim to which, after a few minutes, a glass slide was placed in contact with the surface of the liquid, allowed to remain a short time, and then placed under the microscope. Since its first description, the Willis method has been suggested with many minor adaptations.