Evaluation of heating and liming treatments in sand samples artificially contaminated with Ancylostoma spp. eggs.

Ancylostoma spp. are found worldwide. Infected dog and cat feces can contaminate soil in public places. Despite prophylactic measures being available, studies on direct remediation of Ancylostoma-contaminated soils are scarce. This study aimed to determine the impact of heat treatment and liming on the viability of Ancylostoma spp. eggs in artificially contaminated sandy soil. Sterilized sand samples were contaminated with Ancylostoma spp. eggs extracted from infected dogs' feces. Samples were heated (trial I) to 70 °C or 80 °C, then sieved after 24 hours (212, 90, 38, and 25 µm). Larval cultures were assessed for larval development following heat treatment. Five quicklime concentrations (trial II; 50, 30, 20, 10 and 5%) were used to treat sand. The effect of liming on larval cultures was assessed by measuring embryonic development. Filariform larvae were exposed to 20% quicklime (25 °C and 37 °C, 20 min). Heat treatment destroys Ancylostoma spp. eggs and prevents in vitro larval development. Liming at 50, 30, and 20% concentrations made embryonic development impossible. However, filariform larvae treated with 20% lime solution retained their motility. Heating at 70 °C and liming at 20% were sufficient to make Ancylostoma spp. egg embryogenesis impossible in experimentally contaminated sand samples.

Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples.

BACKGROUND: Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. METHODS: To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy® PowerMax® Soil Kit) with an optional dilution and/or clean-up (Agencourt® AMPure®) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 104 eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. RESULTS: The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy® PowerMax® Soil Kit, and completed with an additional DNA clean-up step with AMPure® beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T. canis eggs in 10-g sand and 10-g soil samples, respectively. CONCLUSIONS: The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment.

Ocorrência de geohelmintos em areia de locais públicos municipais de Videira e Itá SC, Brasil / Occurrence of parasites in public places of sand municipal Videira and Itá, SC, Brazil

A areia das áreas de lazer presentes em praças públicas pode constituir uma via de transmissão de doenças, com potencial risco para as crianças. Um grande número de espécies que podem ser encontradas na areia são potentes agentes patogênicos. Por este motivo, foi expressa a preocupação de que estes locais possam atuar como reservatórios de doenças. Portanto, este estudo objetiva avaliar a qualidade da areia de praças nas cidades de Videira e Itá SC, sob o ponto de vista parasitológico, utilizando os métodos de Lutz e Rugai. No presente trabalho, através da análise microscópica do material coletado, foi possível observar que 40 % das praças analisadas apresentavam-se contaminadas. Foram encontrados ovos de Ascaris lumbricoides e larvas de ancilostomídeos, indicando que a população que frequenta estes locais apresenta risco de contrair estas parasitoses se não forem tomadas medidas de sanitização e prevenção.

The sand of recreational areas found in public squares can be a route of transmission of disease, with potential risk to children. A large number of species that can be found in the sand are potent pathogenic agents. For this reason, concern has been expressed that these places can act as reservoirs or disease vectors. Therefore, this study aims to evaluate the quality of the sand public squares in the cities of Videira and Ita SC, under the parasitological point of view, using Lutz and Rugai methods. In this study, through the microscopic analysis of the collected material, it was observed that 40% of the analyzed squares had become contaminated. Ascaris lumbricoides and hookworm larvae were found, indicating that the population that frequents these places is at risk of contracting these parasites if not taken measures of sanitation and prevention.