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Background  Digital imaging combined with deep-learning-based computational image analysis is a growing area in medical diagnostics, including parasitology, where a number of automated analytical devices have been developed and are available for use in clinical practice. Methods The performance of Parasight All-in-One (AIO), a second-generation device, was evaluated by comparing it to a well-accepted research method (mini-FLOTAC) and to another commercially available test (Imagyst). Fifty-nine canine and feline infected fecal specimens were quantitatively analyzed by all three methods. Since some samples were positive for more than one parasite, the dataset consisted of 48 specimens positive for Ancylostoma spp., 13 for Toxocara spp. and 23 for Trichuris spp. Results The magnitude of Parasight AIO counts correlated well with those of mini-FLOTAC but not with those of Imagyst. Parasight AIO counted approximately 3.5-fold more ova of Ancylostoma spp. and Trichuris spp. and 4.6-fold more ova of Toxocara spp. than the mini-FLOTAC, and counted 27.9-, 17.1- and 10.2-fold more of these same ova than Imagyst, respectively. These differences translated into differences between the test sensitivities at low egg count levels (< 50 eggs/g), with Parasight AIO > mini-FLOTAC > Imagyst. At higher egg counts Parasight AIO and mini-FLOTAC performed with comparable precision (which was significantly higher that than Imagyst), whereas at lower counts (> 30 eggs/g) Parasight was more precise than both mini-FLOTAC and Imagyst, while the latter two methods did not significantly differ from each other. Conclusions In general, Parasight AIO analyses were both more precise and sensitive than mini-FLOTAC and Imagyst and quantitatively correlated well with mini-FLOTAC. While Parasight AIO produced lower raw counts in eggs-per-gram than mini-FLOTAC, these could be corrected using the data generated from these correlations. Graphical Abstract

Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8 mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples. Graphical ► Utility of a flotation method with the use of a wire loop of 8 mm in diameter was evaluated. ► E. tenella oocysts and Heterakis gallinarum eggs in tubes could be collected at a ratio of 2.00 to 3.08. ► Our results may implicate the application for the estimated OPG/ EPG of the samples as a simple and time saving method.

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. 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. 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. 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.

BACKGROUND: One of the challenges for monitoring helminth control programmes based on preventive chemotherapy is the lack of a copro-parasitological gold–standard method that combines good sensitivity with quantitative performance, low cost, and easy-to-learn technique. The aim of our study was to evaluate and compare, the WHO recommended quantitative diagnostic technique (Kato-Katz) and the Mini-FLOTAC. METHODS: Mini-FLOTAC is an innovative method based on floatation of helminths eggs with two different solutions (FS2 and FS7) using a close system (Fill-FLOTAC) with 5% fixative. Kato-Katz was performed following WHO recommendation. The study was carried out in a rural part of Tanzania, close to Lake Victoria, where the laboratory facilities are fairly scarce, and the basic technique used in the local laboratory (direct smear) was taken as reference standard. RESULTS: 201 children were screened for intestinal helminths and 91% of them were found to be positive. The agreement among the three techniques was calculated with k Cohen coefficient and was fairly good (k = 0.4), although the Mini-FLOTAC results were more sensitive for hookworm (98%) with FS2, and for S.mansoni (90%) with FS7 followed by Kato-Katz (91% and 60% respectively) and direct smear (30% and 10% respectively). A good agreement was found between Mini-FLOTAC and Kato-Katz (k = 0.81) with FS7 (k = 0.76) for hookworm diagnosis and a fairly good one for S.mansoni diagnosis (k = 0.5). For both infections we had a poor agreement between the two quantitative techniques and the direct smear (k<0.3). Kato-Katz diagnosed a higher number of eggs (calculated by arithmetic mean) both for hookworm (455 vs 424 EPG) and for S.mansoni (71 vs 58 EPG) compared with the Mini-FLOTAC, but the differences were not significant (p = 0.4). CONCLUSIONS: Mini-FLOTAC is a promising technique, comparable and as sensitive as the Kato-Katz, which is the recommended method in intestinal helminthology for monitoring helminth control programmes. A comparative advantage of the Mini-FLOTAC is that it comprises of a closed system with preserved samples that both protects the operators and allows subsequent examination of the samples. Further studies are needed to validate the mini-FLOTAC with other quantitative techniques (McMaster) and in different settings where other soil-transmitted helminths are also endemic.

Background Faecal egg counts (FECs) are essential for diagnosing helminth infections and guiding treatment decisions. For camels, no evaluations of coproscopic methods regarding precision, sensitivity and correlation between individual and pooled faecal samples are currently available. Methods Here, 410 camel faecal samples were collected in 2022 from South Darfur State, Sudan, and analysed to compare the semi-quantitative flotation, McMaster and Mini-FLOTAC methods in terms of precision, sensitivity, inter-rater reliability and helminth egg count correlations, as well as the effects of pooling samples. Six samples were used to assess precision for McMaster and Mini-FLOTAC, while the remaining 404 samples were evaluated for sensitivity, inter-rater reliability and egg count correlations. Of these, 80 samples were used in pooling experiments. Results Six analyses of each sample ( n  = 6) using the McMaster and Mini-FLOTAC methods revealed no significant difference in the coefficient of variation between the two. For strongyle eggs, 48.8%, 52.7% and 68.6% were positive for McMaster, semi-quantitative flotation and Mini-FLOTAC, respectively. The sensitivity of the three methods showed only minimal improvement when three egg counts were performed on the same sample. McMaster and Mini-FLOTAC had similar sensitivity for Strongyloides spp. (3.5% frequency), while it was lower for semi-quantitative flotation at 2.5%. Mini-FLOTAC was more sensitive for Moniezia spp., detecting 7.7% of positives compared with 4.5% for semi-quantitative flotation and 2.2% for McMaster. For Trichuris spp., frequencies were 0.3% with Mini-FLOTAC, 0.7% with McMaster and 1.7% with semi-quantitative flotation. Mini-FLOTAC also detected higher strongyle eggs per gram (EPG) of faeces (mean 537.4) compared with McMaster (330.1). More samples exceeded treatment thresholds with Mini-FLOTAC, with 28.5% of animals having EPG ≥ 200 compared with 19.3% for McMaster, while 19.1% showed EPG ≥ 500 with Mini-FLOTAC compared with 12.1% with McMaster. There was no significant correlation between individual and pooled strongyle FECs, as indicated by Pearson correlation coefficients of r  ≥ 0.368 ( P  ≥ 0.113) and Spearman correlation. Conclusions Mini-FLOTAC outperformed semi-quantitative flotation and McMaster in diagnosing helminth infections in camels, offering greater sensitivity and detecting higher EPGs, particularly for strongyles, Strongyloides spp. and Moniezia spp. Thus, treatment decisions based on Mini-FLOTAC EPGs will lead to more treatments. Graphical Abstract

Chicken production has increased over the past decade, resulting in a concomitant rise in the demand for more humane options for poultry products including cage-free, free-range, and organic meat and eggs. These husbandry changes, however, have come hand-in-hand with increased prevalence of Ascaridia galli infection, which can cause clinical disease in chickens as well as the occasional appearance of worms in eggs. Additionally, development of anthelmintic resistance in closely related helminths of turkeys highlights the need for closely monitored anthelmintic treatment programs. Manual faecal egg counts (FECs) can be time-consuming and require specialist training. As such, this study sought to validate an automated FEC system for use in detection and quantification of A. galli eggs in chicken faeces. Automated counts using the Parasight System (PS) were compared to traditional manual McMaster counting for both precision and correlation between methods. Overall, ten repeated counts were performed on twenty individual samples for a total of 200 counts performed for each method. A strong, statistically significant correlation was found between methods (R2 = 0.7879, P < 0.0001), and PS counted more eggs and performed with statistically significant higher precision (P = 0.0391) than manual McMaster counting. This study suggests that PS is a good alternative method for performing A. galli FECs and provides a new tool for use in helminth treatment and control programs in chicken operations.

Background Current methods for obtaining fecal egg counts in horses are often inaccurate and variable depending on the analyst’s skill and experience. Automated digital scanning of fecal sample slides integrated with analysis by an artificial intelligence (AI) algorithm is a viable, emerging alternative that can mitigate operator variation compared to conventional methods in companion animal fecal parasite diagnostics. Vetscan Imagyst is a novel fecal parasite detection system that uploads the scanned image to the cloud where proprietary software analyzes captured images for diagnostic recognition by a deep learning, object detection AI algorithm. The study describes the use and validation of Vetscan Imagyst in equine parasitology. Methods The primary objective of the study was to evaluate the performance of the Vetscan Imagyst system in terms of diagnostic sensitivity and specificity in testing equine fecal samples ( n  = 108) for ova from two parasites that commonly infect horses, strongyles and Parascaris spp., compared to reference assays performed by expert parasitologists using a Mini-FLOTAC technique. Two different fecal flotation solutions were used to prepare the sample slides, NaNO 3 and Sheather’s sugar solution. Results Diagnostic sensitivity of the Vetscan Imagyst algorithm for strongyles versus the manual reference test was 99.2% for samples prepared with NaNO 3 solution and 100.0% for samples prepared with Sheather’s sugar solution. Sensitivity for Parascaris spp. was 88.9% and 99.9%, respectively, for samples prepared with NaNO 3 and Sheather’s sugar solutions. Diagnostic specificity for strongyles was 91.4% and 99.9%, respectively, for samples prepared with NaNO 3 and Sheather’s sugar solutions. Specificity for Parascaris spp. was 93.6% and 99.9%, respectively, for samples prepared with NaNO 3 and Sheather’s sugar solutions. Lin’s concordance correlation coefficients for VETSCAN IMAGYST eggs per gram counts versus those determined by the expert parasitologist were 0.924–0.978 for strongyles and 0.944–0.955 for Parascaris spp., depending on the flotation solution. Conclusions Sensitivity and specificity results for detecting strongyles and Parascaris spp. in equine fecal samples showed that Vetscan Imagyst can consistently provide diagnostic accuracy equivalent to manual evaluations by skilled parasitologists. As an automated method driven by a deep learning AI algorithm, VETSCAN IMAGYST has the potential to avoid variations in analyst characteristics, thus providing more consistent results in a timely manner, in either clinical or laboratory settings. Graphical Abstract

Epidemiological studies typically focus on single-parasite systems, although most hosts harbor multiple parasite species; thus, the potential impacts of co-infection on disease dynamics are only beginning to be recognized. Interactions between macroparasites, such as gastrointestinal nematodes, and microparasites causing diseases like TB, AIDS, and malaria are particularly interesting because co-infection may favor transmission and progression of these improtant diseases. Here we present evidence for strong interactions between gastrointestinal worms and bovine tuberculosis (TB) in free-ranging African buffalo (Syncerus caffer). TB and worms are negatively association at the population, among-herd, and within-herd scales, and this association is not solely the result of demographic heterogeneities in infection. Combining data from 1362 buffalo with simple mechanistic models, we find that both accelerated mortality of co-infected individuals and TB transmission heterogeneity caused by trade-offs in immunity to the two types of parasites likely contribute to observed infection patterns. This study is one of the first to examine the relevance of within-host immunological trade-offs for understanding parasite distribution patterns in natural populations.

The diagnosis of gastrointestinal (GI) strongyle infections in equids is still mainly performed using quantitative coprological techniques, like the McMaster (McM), but more sensitive and precise techniques, like FLOTAC (FL) and Mini-FLOTAC (MF), have been proposed over the past 20 years. The present study aimed to compare the analytical performance of these three methods in the diagnosis of strongyle infections in horses. Between October 2023 and June 2024, 32 fecal samples were processed using the McM, FL and MF techniques to identify strongyles’ eggs, estimate their shedding (eggs per gram of feces, EPG), standard errors, sensitivities, precisions, and perform Spearman’s correlation and Cohen’s kappa analyses. The McM detected a higher shedding (584 ± 179 EPG), in comparison with FL and MF, with both these differences being statistically significant (p < 0.001); FL achieved the highest precision (72%), which differed significantly from McM (p = 0.03). All techniques were positively (rs = 0.92–0.96) and significantly (p < 0.001) correlated and shared substantial (k = 0.67–0.76) and significant (p < 0.001) agreement. The MF achieved the highest diagnostic sensitivity (93%), followed by FL (89%) and McM (85%), although not significantly (p = 0.90). These results suggest the usefulness of implementing FL or MF methods in equine medicine for precise and, in the latter case, quick parasitological diagnosis.

Endemic areas for soil-transmitted helminthiases often lack the tools and trained personnel necessary for point-of-care diagnosis. This study pilots the use of smartphone microscopy and an artificial neural network-based (ANN) object detection application named Kankanet to address those two needs. A smartphone was equipped with a USB Video Class (UVC) microscope attachment and Kankanet, which was trained to recognize eggs of Ascaris lumbricoides, Trichuris trichiura, and hookworm using a dataset of 2,078 images. It was evaluated for interpretive accuracy based on 185 new images. Fecal samples were processed using Kato-Katz (KK), spontaneous sedimentation technique in tube (SSTT), and Merthiolate-Iodine-Formaldehyde (MIF) techniques. UVC imaging and ANN interpretation of these slides was compared to parasitologist interpretation of standard microscopy.Relative to a gold standard defined as any positive result from parasitologist reading of KK, SSTT, and MIF preparations through standard microscopy, parasitologists reading UVC imaging of SSTT achieved a comparable sensitivity (82.9%) and specificity (97.1%) in A. lumbricoides to standard KK interpretation (97.0% sensitivity, 96.0% specificity). The UVC could not accurately image T. trichiura or hookworm. Though Kankanet interpretation was not quite as sensitive as parasitologist interpretation, it still achieved high sensitivity for A. lumbricoides and hookworm (69.6% and 71.4%, respectively). Kankanet showed high sensitivity for T. trichiura in microscope images (100.0%), but low in UVC images (50.0%). The UVC achieved comparable sensitivity to standard microscopy with only A. lumbricoides. With further improvement of image resolution and magnification, UVC shows promise as a point-of-care imaging tool. In addition to smartphone microscopy, ANN-based object detection can be developed as a diagnostic aid. Though trained with a limited dataset, Kankanet accurately interprets both standard microscope and low-quality UVC images. Kankanet may achieve sensitivity comparable to parasitologists with continued expansion of the image database and improvement of machine learning technology.