The effect of analyst training on fecal egg counting variability.

Fecal egg counts (FECs) are essential for veterinary parasite control programs. Recent advances led to the creation of an automated FEC system that performs with increased precision and reduces the need for training of analysts. However, the variability contributed by analysts has not been quantified for FEC methods, nor has the impact of training on analyst performance been quantified. In this study, three untrained analysts performed FECs on the same slides using the modified McMaster (MM), modified Wisconsin (MW), and the automated system with two different algorithms: particle shape analysis (PSA) and machine learning (ML). Samples were screened and separated into negative (no strongylid eggs seen), 1-200 eggs per gram of feces (EPG), 201-500 EPG, 501-1000 EPG, and 1001+ EPG levels, and ten repeated counts were performed for each level and method. Analysts were then formally trained and repeated the study protocol. Between analyst variability (BV), analyst precision (AP), and the proportion of variance contributed by analysts were calculated. Total BV was significantly lower for MM post-training (p = 0.0105). Additionally, AP variability and analyst variance both tended to decrease for the manual MM and MW methods. Overall, MM had the lowest BV both pre- and post-training, although PSA and ML were minimally affected by analyst training. This research illustrates not only how the automated methods could be useful when formal training is unavailable but also how impactful formal training is for traditional manual FEC methods.

Helminth Egg Automatic Detector (HEAD): Improvements in development for digital identification and quantification of helminth eggs and their application online.

Helminths are parasitic worms that constitute a major public health problem. Conventional analytical techniques to evaluate helminth eggs in environmental samples rely on different steps, namely sedimentation, filtration, centrifugation, and flotation, to separate the eggs from a variety of particles and concentrate them in a pellet for direct observation under an optical microscope. To improve this process, a new approach was implemented in which various image processing algorithms were developed and implemented by a Helminth Egg Automatic Detector (HEAD). This allowed identification and quantification of pathogenic helminth eggs of global medical importance and it was found to be useful for relatively clean wastewater samples. After the initial version, two improvements were developed: first, a texture verification process that reduced the number of false positive results; and second, the establishment of the optimal thresholds (morphology and texture) for each helminth egg species. This second implementation, which was found to improve on the results of the former, was developed with the objective of using free software as a platform for the system. This does not require the purchase of a license, unlike the previous version that required a Mathworks® license to run. After an internal statistical verification of the system was carried out, trials in internationally recognized microbiology laboratories were performed with the aim of reinforcing software training and developing a web-based system able to receive images and perform the analysis throughout a web service. Once completed, these improvements represented a useful and cheap tool that could be used by environmental monitoring facilities and laboratories throughout the world; this tool is capable of identifying and quantifying different species of helminth eggs in otherwise difficult environmental samples: wastewater, soil, biosolids, excreta, and sludge, with a sensitivity and specificity for the TensorFlow (TF) model in the web service values of 96.82% and 97.96% respectively. Additionally, in the case of Ascaris, it may even differentiate between fertile and non-fertile eggs.

Criteria for identification of Schistosoma mansoni eggs in faecal sediments prepared with the Helmintex method and stained by ninhydrin.

Helmintex is a sensitive method used for detecting Schistosoma mansoni eggs. Here, we describe the observed frequency of six proposed criteria associated with the identification of S. mansoni eggs prepared with the Helmintex method and stained with ninhydrin. The efficacy of these criteria in classifying S. mansoni eggs when applied in various combinations was also examined. Nine observers registered the presence or absence of 6 different criteria in 100 eggs using a microscope at 100x magnification. Ninhydrin purple, which was frequently observed, was the criterion associated with the lowest inter-observer variability. At least three criteria were associated with a significantly better performance in egg identification. In conclusion, ninhydrin staining and a combination of criteria are recommended for microscope examination of faecal sediments.

What makes a good fecal egg count technique?

The first parasite fecal egg counting techniques were described over 100 years ago, and fecal egg counting remains essential in parasitology research as well as in clinical practice today. Several novel techniques have been introduced and validated in recent years, but this work has also highlighted several current issues in this research field. There is a lack of consensus on which diagnostic parameters to evaluate and how to properly design studies doing so. Furthermore, there is a confusing and sometimes incorrect use of terminology describing performance of fecal egg counting techniques, and it would be helpful to address these. This manuscript reviews qualitative and quantitative diagnostic performance parameters, discusses their relevance for fecal egg counting techniques, and highlights some of the challenges with determining them. Qualitative parameters such as diagnostic sensitivity and specificity may be considered classic diagnostic performance metrics, but they generally only have implications at low egg count levels. The detection limit of a given technique is often referred to as the "analytical sensitivity", but this is misleading as the detection limit is a theoretically derived number, whereas analytical sensitivity is determined experimentally. Thus, the detection limit is not a diagnostic performance parameter and does not inform on the diagnostic sensitivity of a technique. Quantitative performance parameters such as accuracy and precision are highly relevant for describing the performance of fecal egg counting techniques, and precision is arguably the more important of the two. An absolute determination of accuracy can only be achieved by use of samples spiked with known quantities of parasite ova, but spiking does not necessarily mimic the true distribution of eggs within a sample, and accuracy estimates are difficult to reproduce between laboratories. Instead, analysis of samples from naturally infected animals can be used to achieve a relative ranking of techniques according to egg count magnitude. Precision can be estimated in a number of different approaches, but it is important to ensure a relevant representation of egg count levels in the study sample set, as low egg counts tend to associate with lower precision estimates. Coefficients of variation generally provide meaningful measures of precision that are independent of the multiplication factor of the techniques evaluated. Taken together, there is a need for clear guidelines for studies validating fecal egg counting techniques in veterinary parasitology with emphasis on what should be evaluated, how studies could be designed, and how to appropriately analyze the data. Furthermore, there is a clear need for better consensus regarding use of terminology describing the diagnostic performance of fecal egg count techniques.

Diagnostic performance of McMaster, Wisconsin, and automated egg counting techniques for enumeration of equine strongyle eggs in fecal samples.

Fecal egg counts are the cornerstone of equine parasite control programs. Previous work led to the development of an automated, image-analysis-based parasite egg counting system. The system has been further developed to include an automated reagent dispenser unit and a custom camera (CC) unit that generates higher resolution images, as well as a particle shape analysis (PSA) algorithm and machine learning (ML) algorithm. The first aim of this study was to conduct a comprehensive comparison of method precision between the original smartphone (SP) unit with the PSA algorithm, CC/PSA, CC/ML, and the traditional McMaster (MM) and Wisconsin (MW) manual techniques. Additionally, a Bayesian analysis was performed to estimate and compare sensitivity and specificity of all five methods. Feces were collected from horses, screened with triplicate Mini-FLOTAC counts, and placed into five categories: negative (no eggs seen), > 0 - ≤ 200 eggs per gram (EPG), > 200 - ≤ 500 EPG, > 500 - ≤ 1000 EPG, and > 1000 EPG. Ten replicates per horse were analyzed for each technique. Technical variability for samples > 200 EPG was significantly higher for MM than CC/PSA and CC/ML (p <  0.0001). Biological variability for samples> 0 was numerically highest for CC/PSA, but with samples > 200 EPG, MM had a significantly lower CV than MW (p =  0.001), MW had a significantly lower CV than CC/PSA (p <  0.0001), CC/ML had a significantly lower CV than both MW and SP/PSA (p <  0.0001, p =  0.0003), and CC/PSA had a significantly lower CV than CC/SP (p =  0.0115). Sensitivity was> 98 % for all five methods with no significant differences. Specificity, however, was significantly the highest for CC/PSA, followed numerically by SP/PSA, MM, CC/ML, and finally MW. Overall, the automated counting system is a promising new development in equine parasitology. Continued refinement to the counting algorithms will help improve precision and specificity, while additional research in areas such as egg loss, analyst variability at the counting step, and accuracy will help create a complete picture of its impact as a new fecal egg count method.

Reliability of three common fecal egg counting techniques for detecting strongylid and ascarid infections in horses.

The detection and quantification of nematode eggs using fecal egg count techniques have an irreplaceable role in equine parasitic control. The reliability, particularly precision and accuracy, of individual techniques have been described only for strongylid infections. The aim of this study was to compare three fecal egg count techniques used for the detection of the two most common equine nematode infections: strongylid and ascarid. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques were tested on spiked fecal samples with various levels of egg concentration (50, 100, 200, 500, 1000 and 3000 eggs per gram) and naturally infected mixed strongylid-ascarid samples with 30 replicates. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques had precision coefficients of variation of 44.33, 35.64 and 18.25% for the strongylid infection and 62.95, 35.71 and 18.95% for the ascarid infection, and percent accuracies (mean count/number of eggs spiked) of 97.53, 88.39 and 74.18% for the strongylid infection and 65.53, 83.18 and 90.28% for the ascarid infection, respectively. Accuracy depended greatly on the type of nematode, but precision did not. The Mini-FLOTAC technique was more precise than the Simple and Concentration McMaster techniques regardless of nematode type. Simple McMaster was the most accurate technique for detecting strongylid eggs, and Mini-FLOTAC was the most accurate technique for detecting ascarid eggs. Our results indicated that none of the current techniques were universally and sufficiently reliable for the simultaneous quantification of both of these common equine nematodes.

Comparison of methods to detect gastrointestinal parasites in llamas and alpacas.

OBJECTIVE: To compare relative sensitivity and overall yields of various methods of fecal examination for gastrointestinal parasites in llamas and alpacas. DESIGN: Prospective study. SAMPLE POPULATION: Fecal samples from 42 alpacas and 62 llamas. PROCEDURES: Fecal samples were analyzed via direct smear, a modified McMaster technique with sucrose solution or saturated saline (approx 36% NaCl) solution, and a centrifugation-flotation procedure. McMaster flotation chambers were examined 15 and 60 minutes after loading. Centrifugation-flotation samples were examined after 10 and 60 minutes of flotation. The proportions of samples with positive results and concentrations of parasites were compared among methods. RESULTS: The centrifugation-flotation technique yielded more positive results than other methods for all parasites except small coccidia. Longer flotation time increased the proportion of positive results and parasite concentrations for all parasites except Nematodirus spp. Longer time in the McMaster chamber made little difference. By use of the modified McMaster technique, sucrose solution yielded more positive results for Trichuris spp, Eimeria macusaniensis, and strongyles, whereas saline solution yielded more positive results for Nematodirus spp and small coccidia. The saline solution McMaster test yielded more positive results for small coccidia than did most other methods, and the sucrose McMaster technique yielded more positive results for Trichuris spp. CONCLUSIONS AND CLINICAL RELEVANCE: The centrifugation-flotation technique appeared to offer clear advantages in detecting infection with E macusaniensis, Trichuris spp, Nematodirus spp, and capillarids. The saline McMaster technique appeared to offer an advantage in detecting small coccidia.

Modification and optimization of the FECPAKG2 protocol for the detection and quantification of soil-transmitted helminth eggs in human stool.

BACKGROUND: Standard diagnosis of human soil-transmitted helminth (STH) infections is based on the microscopic detection of helminth eggs in stool and supports programmatic decision making in control programs. However, the current standard diagnostic techniques still show a number of limitations. Recently, the FECPAKG2 method was developed to detect helminth infections and asses drug efficacy in sheep or cattle. It includes a device that takes digital images of helminth eggs that have been concentrated into one microscopic field of view and stores these images online for future evaluation. The goal of this study was to introduce a standard operating procedure (SOP) for the detection and quantification of human STH eggs using the FECPAKG2 and to optimize 2 crucial steps of the protocol, namely the sedimentation step (aimed at separating sinking eggs from floating debris) and the accumulation step (aimed at concentrating the eggs by flotation). METHODOLOGY/PRINCIPAL FINDINGS: A total of 55 stool samples from naturally infected children were used from 4 different geographical areas (Ethiopia, Laos, Tanzania and Brazil). The results showed that Trichuris eggs generally moved slower than eggs of the other two STH species during both sedimentation in water in the FECPAKG2 sedimenter as during accumulation in flotation solution in the FECPAKG2 cassettes. The highest number of eggs were present in the slurry of the sedimenter after overnight sedimentation (Ascaris: 95.7%, Trichuris: 89.8% and hookworm: 94.2% of the eggs). A minimum of 24 minutes were needed to ensure the accumulation of at least 80% of the eggs from all three STH species in the FECPAKG2 cassette (Ascaris: 96.1%; Trichuris: 88.2% and hookworm: 87.6%). CONCLUSIONS/SIGNIFICANCE: This study introduces for the first time a SOP for the FECPAKG2 method. Different aspects of the method for diagnosing human STH infections were optimized. Our study forms the basis for a thorough and objective evaluation of the system as a diagnostic tool that could be implemented in STH control programs.

[Establishment and application of quality control system for detection of schistosomiasis in Jiangsu Province II Evaluation of pathogenic detection capacity of county-level personnel].

OBJECTIVE: To evaluate the pathogenic detection capacity of county-level organizations in Jiangsu Province, and to improve the field schistosomiasis detection capacity of professional personnel, thus to provide the technical support for the construction of quality control system of schistosomiasis field detection. METHODS: The eggs of Schistosoma japonicum were obtained from rabbit schistosomiasis models and were produced into suspensions at four different concentrations. The county-level workers were invited to hatch the eggs, and the accuracy, detection error rate and omission rate were compared between the detection results and the standard results. The single-blind method was used in the capacity examination. RESULTS: A total of 560 suspensions were detected by 28 counties (districts, cities), and 283 positive samples and 203 negative samples were detected. The total accuracy was 86.79%, total error rate was 9.38%, and total omission rate was 15.77%. The difference between the detection result and standard result was statistically significant (χ2 = 12.99, P < 0.01) . Twenty out of 28 counties (districts, cities) had omission detections, accounting for 71.43%; and 13 had fault detections, accounting for 46.43%. The error rates of workers from the river marshland, hilly areas, water networks, and lake marshland ranged from 4.55% to 43.75%, and the difference was statistically significant (χ2 = 30.34, P < 0.01). The omission rate ranged from 4.17% to 20.45%, and the difference was not statistically significant (χ2 = 5.09, P = 0.17) . The error rates and omission rates of workers from the transmission control areas and transmission interruption areas were 7.50%, 13.33% and 10.42%, 17.13%, respectively, and the differences were not statistically significant (χ2 = 0.229, 0.575, both P > 0.05) . The error rates and omission rates of workers from the areas with or without at least ten years history of transmission control were 11.81%, 5.00% and 16.67%, 14.17%, respectively, and the differences were not statistically significant (χ2 = 2.804, 2.848, both P > 0.05) . The error rates of workers from the areas with or without at least ten years history of transmission interruption were 11.54% and 10.00%, respectively, and the difference was not statistically significant (χ2 = 0.069, P = 0.792), while the correspondent omission rates were 10.90% and 35.00% respectively, and the difference was statistically significant (χ2 = 17.364, P < 0.01) . CONCLUSIONS: The detection error and omission exist in the schistosomiasis examinations in the county-level organizations in Jiangsu Province, and therefore, the field pathogen detection capacity of the professional personnel needs to be further improved.

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.

Ascaris, co-infection and allergy: the importance of analysis based on immunological variables rather than egg excretion.

The ratio of Ascaris seropositivity to the presence of eggs in the faeces was 2.44 in children residing near Cape Town, South Africa. Similar and larger ratios have previously been described for children and women living in the city. The new finding strengthens the concept that when helminthic infections occur together with non-helminthic diseases, an analysis of the interaction must include the use of disease-related immunological variables and not be based only on egg excretion status. One of the reasons is that many egg-negative people who live where helminthiasis is highly endemic are immunologically activated as a result of transitory non-patent or intermittent patent infection by Ascaris and/or other worms.

Comparison of a manual and an automated method to estimate the number of uterine eggs in anisakid nematodes: to Coulter or not to Coulter. Is that the question?

Studies reporting numbers of eggs in vagina and utero in nematodes often give little information of the technique used for the estimations. This situation hampers comparison among studies, because, so far, differences in estimations provided by different techniques have not been assessed. This note examines whether a manual method based on visual counts in aliquots and an automated method using a Coulter counter yield equivalent estimations of egg numbers in vagina and utero of 3 anisakid nematode species (Anisakis simplex, Pseudoterranova decipiens, and Contracaecum osculatum). The number of eggs from 50 females per nematode species was estimated using both techniques. The automated and manual methods yielded similar egg counts (correlation coefficients >0.9 in the 3 species), but the methods were not always statistically equivalent. The automated method was more precise and seemed less dependent on egg density, whereas the manual method was less time-consuming (contrary to previous perceptions) and less expensive. Despite the higher precision of automated counts, the manual technique seemed to produce similar estimates; thus, it may be particularly useful in developing countries where nematode parasitism is prevalent in humans and domestic animals, but scientific resources are limited.

Interpretation of serum antibody response to Anoplocephala perfoliata in relation to parasite burden and faecal egg count.

REASON FOR PERFORMING STUDY: Increased knowledge is needed to assist in the interpretation of presently available diagnostic techniques for infection by the tapeworm Anoplocephala perfoliata in horses. HYPOTHESIS: The suggested cut-off level of an A. perfoliata specific ELISA may not adequately reflect the actual infection level. Hence, faecal egg counts may be a more useful diagnostic test for individual horses than previously reported. METHODS: Eighty-four horses admitted for slaughter at a Danish abattoir were examined for the presence of A. perfoliata. The number of tapeworms, their stage of development and gross pathological mucosal lesions were recorded and compared with serum antibody responses and faecal egg counts. Faecal egg counts were determined in samples from A. perfoliata infected horses using a semi quantitative centrifugation/flotation technique. Blood samples collected at slaughter were analysed by ELISA to determine serum antibody levels against A. perfoliata 12/13 kDa excretory/secretory antigens. RESULTS: Macroscopically visible tapeworms were detected in 24 (29%) of the horses. The overall sensitivity of the faecal egg count was found to be 0.46; however, if the detection limit was increased to above 20 tapeworms, sensitivity increased to 0.89. There was a correlation of 0.71 between worm burden and egg count. The antibody levels correlated significantly with infection intensity despite a wide variation among horses with similar levels of infection. The optimal cut-off value was determined using receiver operating characteristic analysis. If cut-off was chosen at optical density (OD) = 0.7, sensitivity was 0.68 and specificity 0.71. CONCLUSIONS: Both diagnostic methods were capable of revealing potentially pathogenic infections, with the faecal egg count being more applicable on the individual horse level. POTENTIAL RELEVANCE: In the population of Danish horses investigated the serum ELISA test should be interpreted such that horses in need of anti-Anoplocephala treatment have an OD = 0.7 or above.

Sources of variability in the measurement of Ascaris lumbricoides infection intensity by Kato-Katz and qPCR.

BACKGROUND: Understanding and quantifying the sources and implications of error in the measurement of helminth egg intensity using Kato-Katz (KK) and the newly emerging "gold standard" quantitative polymerase chain reaction (qPCR) technique is necessary for the appropriate design of epidemiological studies, including impact assessments for deworming programs. METHODS: Repeated measurements of Ascaris lumbricoides infection intensity were made from samples collected in western Kenya using the qPCR and KK techniques. These data were combined with data on post-treatment worm expulsions. Random effects regression models were used to quantify the variability associated with different technical and biological factors for qPCR and KK diagnosis. The relative precision of these methods was compared, as was the precision of multiple qPCR replicates. RESULTS: For both KK and qPCR, intensity measurements were largely determined by the identity of the stool donor. Stool donor explained 92.4% of variability in qPCR measurements and 54.5% of observed measurement variance for KK. An additional 39.1% of variance in KK measurements was attributable to having expelled adult A. lumbricoides worms following anthelmintic treatment. For qPCR, the remaining 7.6% of variability was explained by the efficiency of the DNA extraction (2.4%), plate-to-plate variability (0.2%) and other residual factors (5%). Differences in replicate measurements by qPCR were comparatively small. In addition to KK variability based on stool donor infection levels, the slide reader was highly statistically significant, although it only explained 1.4% of the total variation. In a comparison of qPCR and KK variance to mean ratios under ideal conditions, the coefficient of variation was on average 3.6 times larger for KK highlighting increased precision of qPCR. CONCLUSIONS: Person-to-person differences explain the majority of variability in egg intensity measurements by qPCR and KK, with very little additional variability explained by the technical factors associated with the practical implementation of these techniques. qPCR provides approximately 3.6 times more precision in estimating A. lumbricoides egg intensity than KK, and could potentially be made more cost-effective by testing each sample only once without diminishing the power of a study to assess population-level intensity and prevalence.

Rationale for Quality Assurance in Fecal Egg Monitoring of Soil-Transmitted Helminthiasis.

Substantial investment has been made into the once "neglected" tropical disease, soil-transmitted helminthiasis, and into control programs that operate within a framework of mapping baseline disease distribution, measuring the effectiveness of applied interventions, establishing when to cease drug administration, and for posttreatment evaluations. However, critical to each of these stages is the determination of helminth infection. The limitations of traditional microscope-based fecal egg diagnostics have not provided quality assurance in the monitoring of parasite disease and suboptimal treatment regimes provide for the potential development of parasite resistance to anthelmintic drugs. Improved diagnostic and surveillance tools are required to protect therapeutic effectiveness and to maintain funder confidence. Such tools may be on the horizon with emergent technologies that offer potential for enhanced visualization and quality-assured quantitation of helminth eggs.

Faecal Parasitology: Concentration Methodology Needs to be Better Standardised.

AIM: To determine whether variation in the preservative, pore size of the sieve, solvent, centrifugal force and centrifugation time used in the Ridley-Allen Concentration method for examining faecal specimens for parasite stages had any effect on their recovery in faecal specimens. METHODS: A questionnaire was sent to all participants in the UK NEQAS Faecal Parasitology Scheme. The recovery of parasite stages was compared using formalin diluted in water or formalin diluted in saline as the fixative, 3 different pore sizes of sieve, ether or ethyl acetate as a solvent, 7 different centrifugal forces and 6 different centrifugation times according to the methods described by participants completing the questionnaire. RESULTS: The number of parasite stages recovered was higher when formalin diluted in water was used as fixative, a smaller pore size of sieve was used, ethyl acetate along with Triton X 100 was used as a solvent and a centrifugal force of 3,000 rpm for 3 minutes were employed. CONCLUSIONS: This study showed that differences in methodology at various stages of the concentration process affect the recovery of parasites from a faecal specimen and parasites present in small numbers could be missed if the recommended methodology is not followed.

Effect of vacuum packing and temperature on survival and hatching of strongyle eggs in faecal samples.

Strongyle eggs of helminths of livestock usually hatch within a few hours or days after deposition with faeces. This poses a problem when faecal sampling is performed in the field. As oxygen is needed for embryonic development, it is recommended to reduce air supply during transport and refrigerate. The present study therefore investigated the combined effect of vacuum packing and temperature on survival of strongyle eggs and their subsequent ability to hatch and develop into L3. Fresh faecal samples were collected from calves infected with Cooperia oncophora, pigs infected with Oesophagostomum dentatum, and horses infected with Strongylus vulgaris and cyathostomins. The samples were allocated into four treatments: vacuum packing and storage at 5 °C or 20 °C (5 V and 20 V); normal packing in plastic gloves closed with a loose knot and storage at 5 °C or 20 °C (5 N and 20 N). The number of eggs per gram faeces (EPG) was estimated every fourth day until day 28 post set up (p.s.) by a concentration McMaster-method. Larval cultures were prepared on day 0, 12 and 28 p.s. and the larval yield determined. For C. oncophora, the EPG was significantly higher in vacuum packed samples after 28 days as compared to normal storage, regardless of temperature. However, O. dentatum EPG was significantly higher in samples kept at 5 °C as compared to 20 °C, irrespective of packing. For the horse strongyles, vacuum packed samples at 5 °C had a significantly higher EPG compared to the other treatments after 28 days. The highest larval yield of O. dentatum and horse strongyles were obtained from fresh faecal samples, however, if storage is necessary prior to setting up larval cultures O. dentatum should be kept at room temperature (aerobic or anaerobic). However, horse strongyle coprocultures should ideally be set up on the day of collection to ensure maximum yield. Eggs of C. oncophora should be kept vacuum packed at room temperature for the highest larval yield.

Testing storage methods of faecal samples for subsequent measurement of helminth egg numbers in the domestic horse.

Parasite infection status, intensity and resistance have traditionally been quantified via flotation techniques, but the need for immediate analysis following defecation imposes limitations and has led to the use of several faecal storage techniques. However, their effect on nematode egg counts has not been systematically validated and is often generalised across taxa despite evidence of differences between species. Here, we take the domestic horse Equus ferus caballus as a model to examine the impact of commonly used storage techniques on egg recovery: 1) high and low concentrations of ethanol and formalin fixative solutions for up to four weeks and 2) refrigeration (3-5°C) over a two-week period. We found a significant decline in faecal egg counts (FEC) following storage in high and low concentrations of both fixative solutions after two weeks, which stabilised after four weeks, and this pattern was uniform across replicates. FECs remained relatively stable over a week of refrigeration, but declined when refrigeration exceeded 8 days. Prior to FEC analysis, we recommend sample refrigeration for no more than one week. Storage in either fixative solution is sub-optimal for the preservation of nematode eggs, although the uniformity of the decline across samples could hold potential for projective calculation of parasite egg shedding when storage time is effectively controlled for.

Effects of aggregation and sample size on composite faecal egg counts in sheep.

Composite faecal egg counts (FEC) are increasingly used to support strategic anthelmintic treatment decisions in grazing livestock. However, their accuracy as estimators of group mean FEC is affected by the number of individual samples included, how thoroughly they are mixed, and the underlying degree of parasite aggregation between individual hosts. This paper uses a Negative Binomial model for parasite aggregation, and a Poisson model for egg distribution within faecal suspensions, in order to optimise composite FEC protocol for commercial sheep flocks. Our results suggest that faecal egg density in a well-mixed composite sample from 10 sheep (3g of faeces from each), estimated by examination of four independently filled McMaster chambers, is likely to provide an adequate estimate of group mean FEC in the majority of situations. However, extra care is needed in groups of sheep for which high levels of FEC aggregation might be expected. The implications of statistical error in FEC estimates depend on how they are used. The simulation-based approach presented here is a powerful tool for investigating the risks of error in FEC-driven treatment decisions in different situations, as well as for the statistical analysis of parasitological data in general.