Human Risks Due to Acanthamoeba spp. in Grass from Public Parks across Leicester City, England †

: Acanthamoeba spp., an emerging opportunistic human parasite, has been previously detected in open-water systems across Leicester city and surrounding rural areas (Leicestershire, England), suggesting a potential risk for the population. A total of 190 grass samples were collected over a year (June 2017 to August 2018) to study the temporal variation, in and around Leicester city and adjacent rural areas. After appropriate pre-treatment to resuspend potential cysts/amoebas present in the grass avoiding potential soil contamination, DNA was extracted from each pre-concentrated sample using the Fast DNA ® Spin kit. Acanthamoeba spp. were detected using a triplex real-time TaqMan PCR assay in 22 grass samples (11.57%). The seasonal study revealed the following prevalence percentages from summer 2017 to summer 2018: 15.7, 10.5, 13.1, 7.89, and 10.5%. To our knowledge, this is the first report reporting the presence and seasonal distribution of Acanthamoeba spp. in grass across Leicestershire.


Introduction
Acanthamoeba spp., an emerging opportunistic free-living heterotrophic protist that can affect humans [1], are the most predominant amoeba in diverse ecological habitats from an ample range of climatic regions [2].Acanthamoeba's life cycle consists of an active and feeding trophozoite and a dormant double-walled polygonal cyst, which are highly resistant to environmental harsh conditions and can remain viable for many years (for more than twenty years) [2,3].As a result, Acanthamoeba has been found in a myriad of different environments, including in soils, sewage, domestic water supplies, ponds, seawater, and water reservoirs across the world.Moreover, Fatemi et al. [4] had recently identified the presence of the highly pathogenic genotypes of T4, T5, and T9 in garden cress, chives, mint, parsley, and basil collected from municipal public markets.
Owing to the increasing relevance of Acanthamoeba spp. as an emerging human concern in recent years, our group is carrying out a comprehensive environmental monitoring study to identify the presence and circulation of these amoebas in different environmental habitats across Leicester city and surrounding rural areas (Leicestershire, England).We initiated this study because of the reported high incidence of Acanthamoeba keratitis (AK) in the United Kingdom, which is about 15 times that recorded in the United States and 7 times that in the Netherlands [5].These authors have related the high incidence with the presence of water storage tanks, which might promote colonisation of domestic water by these amoebas, although a preliminary study carried out by our group recently has not detected the presence of any human-pathogenic free-living amoebas in the tap water in Leicester [6].However, we have isolated Acanthamoeba spp. in different open water systems across Leicestershire, including the River Soar, which crosses Leicester city and attracts a large number of users [7].Thus, the main aim was to determine the presence, distribution/circulation of this emerging opportunistic pathogen in grass from public parks and recreational areas in and around Leicester city and adjacent rural areas, and to identify potential risks to their citizens and users.

Material and Methods
No ethical approval was required for the described study.A total of 190 grass samples were collected over a year (June 2017 to August 2018), with, specifically, 38 samples collected during 5 seasons.Grass samples were collected by cutting the top portion (5-10 cm) to avoid contamination with the topsoils and stored in 50 mL Falcon tubes.Each sample was washed with 15-20 mL of PBS (1% sterile) during a 24 h agitation to obtain a homogenised supernatant, and any potential cyst or amoeba present was resuspended.Supernatants were collected in sterile 50 mL Falcon tubes and centrifuged at 3000 rpm for 30 min.Pellets were collected and suspended in 5 mL of sterile PBS (1%) for the detection of these amoebas.
DNA was extracted from appropriately pre-concentrated grass samples using the FastDNA ® Spin Kit (MP Biomedicals, Solon, OH, USA), following the manufacturer protocol with the modifications described by Gomes et al. [1].These modifications included the introduction of an additional 1/4 inch ceramic sphere in each Fastprep tube and the lysing cycles were performed in triplicate in the homogeniser FastPrep-24 TM 5G (MP Biomedicals, Solon, OH, USA).DNA materials were then purified with NucleoSpin ® Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany), following the manufacturers' instructions.
The triplex real-time TaqMan PCR assay described by Qvarnstrom et al. [8] was used to molecularly detect the presence of Acanthamoeba spp. in the collected grass samples.Positive controls were used as previously described by our group in Peña-Fernández et al. [9].

Results and Discussion
Acanthamoeba spp.were detected in 22 grass samples (11.57%).The seasonal study revealed the following prevalence percentages: 15.7 (summer 2017), 10.5 (autumn 2017), 13.1 (winter 2017-2018), 7.89 (spring 2018), and 10.5% (summer 2018).The highest frequency of Acanthamoeba spp.(15.7%; 8/38) was determined in summer 2017, meanwhile the lowest prevalence was observed in spring 2018 (7.9%; 3/38).The seasonal variations in the prevalence detected did not show statistical differences.To our knowledge, this is the first report reporting the presence and seasonal distribution of Acanthamoeba spp. in grass across different public parks and recreational green areas in Leicester city and surrounding rural areas in Leicestershire.The moderate to high seasonal prevalence found would be logical, owing to the high environmental resistance of their cysts [1][2][3][4][5].Our results would suggest a potential unknown source for Acanthamoeba spp.In Leicester city/Leicestershire, which could represent a serious hazard to human health that should be appropriately tackled, to prevent and minimise the exposure to the users of these public and recreational areas in the East Midlands region explored.
Although data on the presence and/or prevalence of Acanthamoeba spp.And other free-living amoebas in grass are limited in the literature, different recent studies have reported their presence in a wide range of vegetables, including lettuce, carrots, cauliflower, radishes, onions, spinach, and tomatoes [4], which might explain their presence found in the grass.
The presence and seasonal circulation of Acanthamoeba spp.Found in the grass could be explained by their presence in topsoils.However, and although our group has detected their presence in some topsoils sampled in a pilot study carried out on different public parks in Leicester, further monitoring studies would be needed to fully understand the potential role of the (top)soils on the presence and distribution of Acanthamoeba spp.Found in the grass samples.Moreover, although their interactions with domestic and wild animals are not well-understood, a potential zoonotic reservoir might explain the moderate to high distribution found in a similar fashion as for other parasites.Thus, although limited, Acanthamoeba species have been found in domestic and wild animals [10].

Conclusions
This is the first report reporting the presence and seasonal distribution of Acanthamoeba spp.In grass collected from public parks/recreational areas in Leicester city and in different rural areas across Leicestershire, suggesting a moderate to high seasonal prevalence, which highlights a concern for public health.Leicestershire's citizens could be exposed when playing sports, or by contact of the cornea or tissue wounds with the grass.Further genotyping studies are required for a better characterisation of the distribution and circulation of Acanthamoeba spp.In Leicestershire, and to identify appropriate public health interventions and decontamination techniques to minimise the risks identified, especially to protect individuals with their immune systems compromised.