World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine.

The faecal egg count reduction test (FECRT) remains the method of choice for establishing the efficacy of anthelmintic compounds in the field, including the diagnosis of anthelmintic resistance. We present a guideline for improving the standardization and performance of the FECRT that has four sections. In the first section, we address the major issues relevant to experimental design, choice of faecal egg count (FEC) method, statistical analysis, and interpretation of the FECRT results. In the second section, we make a series of general recommendations that are applicable across all animals addressed in this guideline. In the third section, we provide separate guidance details for cattle, small ruminants (sheep and goats), horses and pigs to address the issues that are specific to the different animal types. Finally, we provide overviews of the specific details required to conduct an FECRT for each of the different host species. To address the issues of statistical power vs. practicality, we also provide two separate options for each animal species; (i) a version designed to detect small changes in efficacy that is intended for use in scientific studies, and (ii) a less resource-intensive version intended for routine use by veterinarians and livestock owners to detect larger changes in efficacy. Compared to the previous FECRT recommendations, four important differences are noted. First, it is now generally recommended to perform the FECRT based on pre- and post-treatment FEC of the same animals (paired study design), rather than on post-treatment FEC of both treated and untreated (control) animals (unpaired study design). Second, instead of requiring a minimum mean FEC (expressed in eggs per gram (EPG)) of the group to be tested, the new requirement is for a minimum total number of eggs to be counted under the microscope (cumulative number of eggs counted before the application of a conversion factor). Third, we provide flexibility in the required size of the treatment group by presenting three separate options that depend on the (expected) number of eggs counted. Finally, these guidelines address all major livestock species, and the thresholds for defining reduced efficacy are adapted and aligned to host species, anthelmintic drug and parasite species. In conclusion, these new guidelines provide improved methodology and standardization of the FECRT for all major livestock species.

How to improve the standardization and the diagnostic performance of the fecal egg count reduction test?

Although various studies have provided novel insights into how to best design, analyze and interpret a fecal egg count reduction test (FECRT), it is still not straightforward to provide guidance that allows improving both the standardization and the analytical performance of the FECRT across a variety of both animal and nematode species. For example, it has been suggested to recommend a minimum number of eggs to be counted under the microscope (not eggs per gram of feces), but we lack the evidence to recommend any number of eggs that would allow a reliable assessment of drug efficacy. Other aspects that need further research are the methodology of calculating uncertainty intervals (UIs; confidence intervals in case of frequentist methods and credible intervals in case of Bayesian methods) and the criteria of classifying drug efficacy into 'normal', 'suspected' and 'reduced'. The aim of this study is to provide complementary insights into the current knowledge, and to ultimately provide guidance in the development of new standardized guidelines for the FECRT. First, data were generated using a simulation in which the 'true' drug efficacy (TDE) was evaluated by the FECRT under varying scenarios of sample size, analytic sensitivity of the diagnostic technique, and level of both intensity and aggregation of egg excretion. Second, the obtained data were analyzed with the aim (i) to verify which classification criteria allow for reliable detection of reduced drug efficacy, (ii) to identify the UI methodology that yields the most reliable assessment of drug efficacy (coverage of TDE) and detection of reduced drug efficacy, and (iii) to determine the required sample size and number of eggs counted under the microscope that optimizes the detection of reduced efficacy. Our results confirm that the currently recommended criteria for classifying drug efficacy are the most appropriate. Additionally, the UI methodologies we tested varied in coverage and ability to detect reduced drug efficacy, thus a combination of UI methodologies is recommended to assess the uncertainty across all scenarios of drug efficacy estimates. Finally, based on our model estimates we were able to determine the required number of eggs to count for each sample size, enabling investigators to optimize the probability of correctly classifying a theoretical TDE while minimizing both financial and technical resources.

Application of a Poisson distribution quality control measure to the analysis of two human hookworm drug treatment studies in Ghana.

We examined faecal egg count reduction tests (FECRTs) conducted with hookworm-infected humans in Ghana in 2007 (study 1) and 2010 (study 2) in order to explore aspects of the test analysis. Some subjects showed increased FEC following drug treatment. This occurred mostly in <150 epg pre-treatment FEC subjects. We sought a means to remove 'erroneous' negative drug efficacy cases from the FECRT analysis. Pre- and post-treatment FECs from negative drug efficacy cases were examined to determine whether they represented replicates from a single randomly distributed sample, that is, if they were consistent with a Poisson distribution. Cases where the post-treatment FEC was greater than that expected if it and the pre-treatment sample had been taken from a single random distribution of eggs were excluded from the FECRT. We suggest that these cases most likely represent non-random distribution of eggs in stools, day-to-day variations in egg excretion, or worm patency onset after drug treatment, and hence are not accurate measurements of drug efficacy. This led to exclusion of the most extreme negative drug efficacy cases, with significant increases in overall drug efficacy for study 1 (81.6% vs 89.2%) and study 2 (86.7% vs 89.4%). Excluding FEC <150 individuals from the analysis also increased the study 1 efficacy (81.6% vs 88.9%), however, this resulted in the exclusion of 45% of the study subjects, compared to the exclusion of just 5% using the Poisson distribution method. While low FEC subjects are excluded from livestock FECRTs, the significant prevalence of such subjects in human FECRTs suggests that their exclusion may not be practical. Hence, we suggest that the influence of low FECs can be minimised by excluding 'erroneous' negative efficacy cases using a simple Poisson distribution analysis.

Novel insights in the fecal egg count reduction test for monitoring drug efficacy against soil-transmitted helminths in large-scale treatment programs.

BACKGROUND: The fecal egg count reduction test (FECRT) is recommended to monitor drug efficacy against soil-transmitted helminths (STHs) in public health. However, the impact of factors inherent to study design (sample size and detection limit of the fecal egg count (FEC) method) and host-parasite interactions (mean baseline FEC and aggregation of FEC across host population) on the reliability of FECRT is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: A simulation study was performed in which FECRT was assessed under varying conditions of the aforementioned factors. Classification trees were built to explore critical values for these factors required to obtain conclusive FECRT results. The outcome of this analysis was subsequently validated on five efficacy trials across Africa, Asia, and Latin America. Unsatisfactory (<85.0%) sensitivity and specificity results to detect reduced efficacy were found if sample sizes were small (<10) or if sample sizes were moderate (10-49) combined with highly aggregated FEC (k<0.25). FECRT remained inconclusive under any evaluated condition for drug efficacies ranging from 87.5% to 92.5% for a reduced-efficacy-threshold of 90% and from 92.5% to 97.5% for a threshold of 95%. The most discriminatory study design required 200 subjects independent of STH status (including subjects who are not excreting eggs). For this sample size, the detection limit of the FEC method and the level of aggregation of the FEC did not affect the interpretation of the FECRT. Only for a threshold of 90%, mean baseline FEC <150 eggs per gram of stool led to a reduced discriminatory power. CONCLUSIONS/SIGNIFICANCE: This study confirms that the interpretation of FECRT is affected by a complex interplay of factors inherent to both study design and host-parasite interactions. The results also highlight that revision of the current World Health Organization guidelines to monitor drug efficacy is indicated. We, therefore, propose novel guidelines to support future monitoring programs.

Associations between trichostrongylid worm egg count and productivity measures in Dorper lambs.

Two flocks of pure bred Dorper lambs were managed separately according to sex (283 ewe lambs and 212 ram lambs) in southern Western Australia. Faecal sample collection, weighing and body condition assessments were performed for each lamb on 2 occasions, specifically pre-weaning (approximately 14 weeks of age) and post-weaning (approximately 9 months of age). Body condition score (BCS) was assessed using a scale of 1 (very thin, emaciated) to 5 (excessively fat). Faecal worm egg counts (WECs) were measured using a modified McMaster technique and larval cultures were performed to identify trichostrongylid nematode genera present. Eye muscle and c-site fat depths were measured using ultrasound at post-weaning. Lambs received an abamectin anthelmintic treatment at weaning (18 weeks of age). Worm egg count data was assessed for normality of data distribution and homogeneity of variance. This data was transformed using log(10)(WEC+25) to stabilise variances between groups prior to statistical analyses and general linear models were used to assess relationships between WEC and productivity measures. Mean WECs were 564 eggs per gram of faeces (EPG) and 514 EPG at pre- and post-weaning in the ewe flock and 552 EPG and 480 EPG at pre- and post-weaning in the ram flock. Teladorsagia (Ostertagia) circumcincta, Trichostrongylus spp. and Oesophagostomum spp. larvae were identified. No lambs with WEC<500 EPG pre-weaning had WEC>1000 EPG post-weaning. Ewe and ram lambs with WEC>1000 EPG at pre-weaning were 42 (12-145 95% CI) and 37 (9-153) times more likely to have WEC>1000 EPG at post-weaning than lambs with WEC 501-1000 EPG at pre-weaning. There were no significant relationships between WEC and live weight in the ram flock, while relationships between WEC and live weight were inconsistent in the ewe lamb flock. There was no relationship between WEC and eye muscle or c-site fat depth. Significant negative relationships between WEC and BCS were identified at pre- and post-weaning for both flocks. Lambs with WEC<500 EPG had 0.19-0.61 higher mean BCS than lambs with WEC>1000 EPG at pre- and post-weaning. In conclusion, high WEC was associated with lower body condition in Dorper lambs, however the relationship between WEC and live weight was inconsistent and there was no effect on eye muscle depth.

Vaccination reduces the viral load and the risk of transmission of Jembrana disease virus in Bali cattle.

The efficacy of a tissue-derived vaccine, which is currently used in Indonesia to control the spread of Jembrana disease in Bali cattle, was determined by quantifying the viral load in plasma following experimental infection with Jembrana disease virus. Virus transmission is most likely to occur during the acute phase of infection when viral titers are greater than 10(6) genomes/ml. Vaccinated cattle were found to have a 96% reduction in viral load above this threshold compared to control cattle. This would reduce the chance of virus transmission as the number of days above the threshold in the vaccinated cattle was reduced by 33%. Viral loads at the onset and resolution of fever were significantly lower in the vaccinated cattle and immune function was maintained with the development of antibody responses to Env proteins within 10-24 days post challenge. There was, however, no significant reduction in the duration of the febrile period in vaccinated animals. The duration and severity of clinical parameters were found to be variable within each group of cattle but the quantification of viral load revealed the benefits of vaccinating to reduce the risk of virus transmission as well as to ameliorate disease.

High-level ivermectin resistance in a field isolate of Haemonchus contortus associated with a low level of resistance in the larval stage: implications for resistance detection.

The IVPro isolate of Haemonchus contortus was isolated in 1999 after significant numbers of the parasite survived an ivermectin capsule treatment of grazing sheep acquiring a natural infection at Prospect, NSW, Australia. The isolate shows high-level resistance to ivermectin (faecal egg count is unaffected by ivermectin oral treatment at 0.2mg kg(-1)). The larval LC(50), as assessed by larval development assays (LDAs), is only approximately two-fold higher than several susceptible isolates, making it difficult to detect the resistance using larval LC(50) as an indicator. However, the isolate shows the presence of a small proportion of the population (<20%) able to develop at significantly higher drug concentrations than the susceptible isolates. Hence, if the IVPro and susceptible isolates are compared at the LC(99) level, the IVPro isolate is readily identifiable as resistant. This degree of distinction at the LC(99) allows the IVPro isolate to be identified as resistant by simply observing the highest drug concentration in the development assay at which some larvae develop relative to the susceptible isolates. Assessing the development assay using this criterion allows a distinction between IVPro and the susceptible isolates equivalent to 10-fold differences in drug concentration, greatly increasing the likelihood of detecting the resistant isolate in routine resistance tests. This study highlights the need to examine this aspect of LDAs in order to detect the type of resistance displayed by IVPro H. contortus.