NOVEMBER 2022
589
areflection of the proportion of eggs lost during the mixing, filtering and flotation steps of the procedure. All techniques
will perform with some degree of egg loss, and the larger this is, the lower the accuracy will be. While using an accurate technique may sound important, it has relatively limited implications for faecal egg counts, for several reasons. First of all, the true egg count is never known, so accuracy cannot be truly estimated. Secondly, since egg counts do not correlate with worm counts, there is little to no diagnostic value in the magnitude of the count. As long as the same technique is used pre- and post-treatment, accuracy will not affect the FECRT at all, since the result is expressed as the percent change between pre- and post-treatment egg counts. However, accuracy could interfere with classification of horses into low, moderate and high strongylid shedders. For example, Stoll and Wisconsin based techniques tend to perform with lower accuracy and generate much lower counts than McMaster (Cain et al., 2020, 2021), and this has caused confusion among veterinarians in the past. However, the thresholds used to identify low, moderate and high strongyle shedders could be adjusted if a technique with a lower accuracy is used. Precision is a measure of the variation between repeated
counts on the same sample (Fig 1), and is synonymous with repeatability. Many veterinarians and horse owners have experienced frustrations over the sometimes large variations observed between egg counts determined from the same horse over a short amount of time. While some of this variation is due to biological variability in the samples (see the section entitled Egg count variability), some egg counting techniques produce more variable results than others. For example, we have shown McMaster techniques to be more precise than Wisconsin as measured by lower coefficients of variation (Cain et al., 2020). The precision of a given technique has considerable implications, as it will affect anthelmintic efficacy estimates determined with the FECRT. It is important to be able to reliably distinguish between a true reduction of efficacy and random variation of pre- and post- treatment egg counts. Using a technique documented to perform with higher precision will help ensure a more reliable FECRT result. Furthermore, when egg counts are determined to identify low, moderate and high strongyle egg shedders, precision has implications for classifying horses as being above or below the chosen thresholds. Thus, interpretation of results is largely affected by precision, which should be considered when choosing egg counting technique and interpreting the results. We have found that the most precise faecal egg counting techniques to be performing with coefficients of variation below 20%, whereas less precise techniques can be 50% or higher (Cain et al., 2020; Noel et al., 2017; Scare et al., 2017). Among manual egg counting techniques currently available for equine practitioners, the Mini-FLOTAC technique has been shown to be the most precise (Noel et al., 2017). McMaster has moderate precision (Noel et al., 2017; Cain et al., 2020), while a test tube and coverslip-based technique such as Wisconsin performs with a lower level of precision (Cain et al., 2020). It is important to recognise that the performance of a
manual faecal egg counting technique is highly dependent on the personnel doing the counts. Even the most accurate and precise technique will perform very poorly, if the operators have not been trained properly. We recently evaluated the performance of four different faecal egg
counting techniques in the hands of three untrained and inexperienced analysts. We had them complete the same study protocol both before and after formal laboratory training, and we demonstrated that precision improved significantly after the training (Cain et al., 2021). We also found that the Wisconsin technique performed with the lowest accuracy, but that this also improved after the training (Cain et al., 2021). Thus, veterinarians offering egg counts in- house should ensure adequate training of their personnel and regularly monitor egg count performance for verification of diagnostic quality. Furthermore, veterinarians using diagnostic laboratories for faecal egg count services should request documentation of technique performance and protocols for diagnostic test quality assurance amongst laboratory operators.
Which technique to use?
The multitude of available faecal egg counting techniques and modifications of these can be challenging to manoeuvre, and it can be difficult to find a clear answer to the simple question of which technique is the most appropriate to use in equine veterinary practice. Table 1 summarises accuracy and precision levels for three commonly used manual techniques; McMaster, Mini-FLOTAC and Wisconsin. In order to make the most appropriate choice, veterinarians should consider the intended use of the egg count results. As outlined herein, the three primary applications of faecal egg counts in equine practice are 1) anthelmintic efficacy screening, 2) monitoring of strongylid egg shedding levels and 3) monitoring of ascarid presence. Below, some considerations are suggested for each of these.
Anthelmintic efficacy screening A technique with high precision is preferred. In addition, the reliability of the efficacy estimate is increasing with more eggs being counted. Thus, a technique that counts more eggs under the microscope before converting to eggs per gram is preferred. Of the techniques included in Table 1, the Mini- FLOTAC would be the best choice.
Monitoring of strongylid shedding levels A relatively precise and accurate technique is preferred. Techniques with lower accuracy can be employed, but classification thresholds may need adjustment since egg count magnitude will be lower. Of the techniques included in Table 1, the McMaster would be a sufficient choice, but Mini- FLOTAC would obviously be better.
Monitoring of ascarid presence Monitoring for ascarids in weanlings, yearlings and youngsters is primarily a qualitative exercise, that is, checking for presence or absence of ascarid eggs. Thus, the quantitative performance matters less for this purpose, but the diagnostic sensitivity is of importance. The challenge is that diagnostic sensitivities for detection of ascarid eggs in faecal samples have not been estimated for a wide range of techniques. A test tube and cover slip technique (Stoll) was found to have a diagnostic sensitivity of 72% for detection of ascarid infection (Nielsen et al., 2010), but estimates do not exist for counting chamber-based techniques. Extrapolation from performance with strongylid egg counts should be made with the greatest caution as it cannot be assumed that diagnostic
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