Monitoring drug efficacy against gastrointestinal nematodes when faecal egg counts are low: do the analytic sensitivity and the formula matter?
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- Levecke, B., Rinaldi, L., Charlier, J. et al. Parasitol Res (2011) 109: 953. doi:10.1007/s00436-011-2338-z
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The faecal egg count reduction test (FECR) is the recommended technique to monitor anthelmintic drug efficacy in livestock. However, results are often inconclusive due to the low analytic sensitivity of the diagnostic technique or the conflict in results from FECR formulae. A novel experimental set-up was, therefore, used to compare the impact of analytic sensitivity and formulae on FECR results. Four McMaster techniques (analytic sensitivities 50, 33.3, 15 and 10) and a FLOTAC technique (analytic sensitivity ~ 1) were used on faecal samples of 30 calves with a FEC of less than 200 eggs per gram. True drug efficacies of 70%, 80% and 90% were experimentally mimicked by comparing FEC before and after dilution (3:10, 2:10 and 1:10, respectively). The FECR was summarized using group (FECR(1)) and individual (FECR(2)) based formulae. There was a significant increase in precision of FECR when the analytic sensitivity increased (p < 0.0001). The precision also depended on the formula used, FECR(1) (p < 0.05) resulting in more precise FECR compared to FECR(2). The accuracy of the FECR differed marginally between the two formulae (p = 0.06), FECR(1) being more accurate. In conclusion, the present study describes a novel methodology to compare techniques for the precision and the accuracy of their FECR results. The results underscored that techniques with high analytic sensitivity will improve the interpretation of FECR in animal populations where baseline FEC are low. They also point out that the precision of individual-based formulae is affected by the analytic sensitivity.
At present, the McMaster techniques (Ministry of Agriculture, Fisheries and Food 1986) are recommended by the World Association for the Advancement of Veterinary Parasitology to monitor drug efficacy against gastrointestinal nematodes in livestock based on the reduction in faecal egg counts (FECR) (Coles et al. 1992). Although they are simple and user friendly (Levecke et al. 2009), their chief limitation is the high analytic sensitivity (ranging from 10 to 50) that may thwart interpretation of the obtained FECR (El-Abdellati et al. 2010). This is particularly so in small herds, when the parasite population is highly aggregated or when animals are excreting a low number of eggs. With the development of novel faecal egg count (FEC) techniques with an analytic sensitivity of 1, such as the FLOTAC (Cringoli et al. 2010), it has been proposed that their use is likely to result in more precise and accurate estimates of the true FECR. However, to date, there are no studies available comparing FEC techniques for monitoring drug efficacy based on the FECR.
Apart from detection techniques, important differences in FECR have been observed depending on the formula used to calculate the reduction, individual-based formulae resulting in lower FECR compared to group-based formulae (Cabaret and Berrag 2004; Vercruysse et al. 2011). However, it was noted that these differences disappeared when only subjects with high FEC were considered, suggesting that the recommended formula to use may depend on the analytic sensitivity and/or the baseline FECs of the subjects under study.
Until now, effects of formulae have been studied by simulation studies (Dobson et al. 2009), as this allows to compare the FECR with a fixed true drug efficacy. However, the results of these studies depend strongly on the assumptions made, particularly for the FEC obtained by the techniques. The observed FEC are modelled according to the Poisson distribution, which implies that techniques with the same analytic sensitivities result in comparable FEC results. Yet, as illustrated by Levecke et al. 2009, this is not always the case.
In the present study, a novel experimental set-up which allowed to determine the true drug efficacy was used to compare detection techniques with different analytic sensitivities for FECR results when baseline FEC are low. In addition, differences in individual- and group-based formulae on FECR were examined.
Materials and methods
Fresh faecal samples were collected from 30 calves excreting a low number of FEC (<200 eggs per gram faeces (EPG), based on FLOTAC). The true drug efficacy (TDE) resulting in faecal egg count reduction (FECR) of 70%, 80% and 90% was experimentally mimicked by comparing baseline FEC with FEC after dilution of the baseline samples (3:10, 2:10 and 1:10). To this end, samples were processed as follows:
For each sample, 10 g was homogenized in tap water (90 ml). The suspension was sieved three times to discard large debris; subsequently, tap water was added to the residue (containing the eggs) up to a volume of 100 ml, resulting in a final concentration of 1 g faeces in 10 ml. For each detection technique, aliquots of 1, 2, 3 and 10 ml of this stock were transferred to centrifuge tubes, representing FEC for a TDE of 90%, 80% and 70%, and baseline FEC. Subsequently, the tubes were centrifuged for 3 min at 170×g, and the supernatant was discarded.
The total volume in which aliquots are re-suspended, the volume examined and the analytic sensitivity for the McMaster (four variants) and the FLOTAC techniques
Total volume (ml)
Volume examined (ml)
Analytic sensitivity (= total volume/volume examined
The baseline FEC (mean, minimum [min.], maximum [max.]) and the number of samples with FEC of zero for the different analytic sensitivities
Number samples with FEC = 0
Figure 2 summarizes the bias of the FECR and the 95% confidence intervals for the three dilutions across the different analytic sensitivities and formulae, indicating that 10 out of the 30 FECRT results were biased (95% CI did include the zero). The highest analytic sensitivity (6/10) systematically overestimated the true drug efficacy (bias < 0), whereas the remaining analytic sensitivities (4/10) also resulted in underestimations (bias > 0).
The present study assessed the impact of the analytic sensitivity of the technique and the formula on the precision and accuracy for FECR results when baseline FEC are low.
The results indicated that an increase in analytic sensitivity and a group-based formula (FECR(1)) results in more precise FECR, improving the final interpretation. The effect of analytic sensitivity is not surprising, since a difference in one egg count after treatment can have a considerable impact on the FECR for techniques with a high analytic sensitivity. The difference in formulae is caused by the large number of animals with a FEC of zero at baseline, resulting in an increase in variation for FECR(2), where these animals ought to be excluded, but not for FECR(1). This also clarifies why the formulae do not affect the precision of the lowest analytic sensitivity (~1), because for this analytic sensitivity, baseline FEC were available for all animals.
For the accuracy of the FECR results, only a marginal difference was found between the two formulae, the group-based formula (FECR(1)) resulting in more accurate FECR (approached a bias of zero) compared to the individually based formula (FECR(2)). Yet, this finding was more pronounced for low analytic sensitivities, which can also be explained by the two reasons mentioned above. The discrepancy in formulae confirms previous studies involving sheep (Cabaret and Berrag 2004) and humans (Vercruysse et al. 2011) where FECR(2) based on the McMaster technique (multiplication 5–50) yielded lower drug efficacies compared to FECR(1), and indicate that the use of FECR(2) to summarize FECR is not recommended for techniques with low analytic sensitivities when baseline FEC are low.
For the analytic sensitivity of 1, there was a systematic decrease in bias with increasing TDE. This observation is unexpected but may be due to the experimental set-up. Because the ratio of the volume of faeces to volume of flotation solution increased with decreasing drug efficacy, the density of the flotation solution may have been lower in samples where low drug efficacy was mimicked, resulting in lower FEC and rendering a positive bias.
This study focused on the impact of formula and analytic sensitivity; however, other factors inherent to the study design (e.g. inclusion of an untreated control group and sample size) or host–parasite interaction resulting in aggregated FEC both across and within hosts should not be ignored. For instance, the amount of faeces homogenized prior to the examination (McMaster, 3–4 g; FLOTAC, 10 g) may already thwart FEC, as the eggs are not equally distributed among faecal samples.
In conclusion, we used a novel methodology to assess the precision and the accuracy of FECR results based on different analytic sensitivities and formulae. The comparison indicated that techniques with a high analytic sensitivity are preferred for monitoring drug efficacy in populations with low baseline FEC. They also point out that the interpretation of individual-based formulae is affected by the analytic sensitivity of the technique. Finally, further studies are needed to improve the interpretation of FEC considering other factors such as the inclusion of an untreated control group, sample size and aggregation of the FEC across and within hosts.
BL is funded by the Fund for Scientific Research–Flanders (Belgium) (F.W.O.–Vlaanderen). The research of J.C. is supported by the Agency of Innovation by Science and Technology (IWT Vlaanderen, projectOZM090697).