Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati.

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15­120 min), and different concentrations of malachite green dye (0.004­0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10­100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.

Environmental Toxocara spp. presence in crowded squares and public parks from San Juan Province, Argentina: A call for a "One Health" approach.

Introduction: Canine soil-transmitted helminth (cSTH) parasites need specific environmental conditions to complete their life cycle. Toxocara canis and T. cati are the most important zoonotic cSTH, since they are the causal agents of human toxocariasis. Canine STHs are dispersed in feces from infected domestic and wildlife canines. In this study, the presence of STH in canine feces was evaluated in 34 crowded public parks and squares from San Juan Province (Argentina). Methods: Fecal samples were collected during different seasons in 2021-2022 and analyzed by standard coprological methods, including Sheather and Willis flotation and Telemann sedimentation. InfoStat 2020, OpenEpi V. 3.01 and R and RStudio® were used for statistical analysis and QGIS 3.16.10 for mapping. Results: From a total of 1,121 samples collected, 100 (8.9%) were positive for at least one intestinal parasite (IP) and three cSTH species were detected: Toxocara spp., Toxascaris leonina and Trichuris vulpis. The most prevalent cSTH species was T. vulpis (64/1121; 0.057%), while the least prevalent was Toxocara spp. (19/1121; 0.017%). The detection of Toxocara spp. eggs was significantly different depending on the season. The geo-spatial variation of each cSTH per season is described. Discussion: This is the first study in San Juan Province to identify environmental contamination of cSTHs in public areas. The specific localization of areas with the presence of cSTH eggs could provide information to guide strategies to reduce the cSTH infection burden in dogs and promote serological screening of the human population for Toxocara spp. Given the zoonotic nature of Toxocara spp. We hope this information will help to reinforce activities of control programs, focusing on the "One Health" approach.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples.

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

Development of a copro-LAMP assay for detection of several species of Echinococcus granulosus sensu lato complex.

Cystic echinococcosis represents a significant problem in human and animal health and constitutes one of the most severe Neglected Tropical Diseases prioritized by the World Health Organization. The etiological agent is the complex Echinococcus granulosus sensu lato (s. l.), composed of several species/genotypes. Diagnosis in the definitive host and molecular epidemiology studies are important points for cystic echinococcosis control. Here we developed a new copro-LAMP assay, LAMP EGSL, for diagnosis in the definitive host for simultaneous detection of Echinococcus granulosus sensu stricto (s. s.), Echinococcus ortleppi, and Echinococcus canadensis species. Also, the analytical sensitivity, specificity and plausibility of performance in a rural context of a previously reported species-specific LAMP reaction, was evaluated. Both reactions showed high analytical sensitivity values (10 fg-100 fg DNA) and did not show cross reaction with DNA from host or other helminthic parasites. LAMP EGSL was performed with samples from an endemic area. In addition, the alkaline hydrolysis of one E. granulosus s. s. adult parasite followed by specific LAMP to E. granulosus s. s. was performed in a laboratory with low resources from another cystic echinococcosis endemic area. The results obtained suggest that LAMP EGSL represents a potential tool for canine diagnosis that could be useful for cystic echinococcosis control programs. In addition, we showed that LAMP reaction for E. granulous s. s., E. ortleppi and E. canadensis specific detection, could be useful for molecular epidemiology studies applicable to the definitive host. Both reactions were performed in endemic, rural areas without sophisticated equipment.