Optimized DNA-based Identification of Toxocara spp. Egg in Difficult Matrices: A Case for Specific and Sensitive Identification of Geohelminth Eggs

Optimized DNA-based Identification of Toxocara spp. Egg in Difficult Matrices: A Case for Specific and Sensitive Identification of Geohelminth Eggs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimized DNA-based Identification of Toxocara spp. Egg in Difficult Matrices: A Case for Specific and Sensitive Identification of Geohelminth Eggs Wojciech Jarosz, Jean-François Durant, Leonid Mwana wa bene Irenge, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-264679/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Toxocara canis ( T. canis ) and Toxocara cati ( T. cati ) are worldwide-distributed roundworms of canids and felids and causative agents of human toxocarosis, via ingestion of Toxocara eggs disseminated in the environment. Control of Toxocara infections is constrained by the lack of sensitive methods for screening of animal feces and environmental samples potentially contaminated by Toxocara eggs. We previously developed a quantitative duplex real-time PCR (qPCR) for sensitive and specific detection of T. canis and T. cati . In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs present in environmental samples was set up. For this purpose, the influence of different methods for eggs lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. Methods Soil and sand (10g) samples were spiked with egg suspensions of T. canis . DNA was extracted from Toxocara eggs, using different DNA extraction kits (FastDNA™ SPIN Kit for Soil and DNeasy® PowerMax® Soil Kit), and an additional clean-up step (Agencourt® AMPure®). The efficiency of the above-developed process was compared with the conventional flotation-centrifugation and observation of Toxocara eggs under light microscopy. Results The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, DNA extraction with the DNeasy® PowerMax® Soil Kit and an additional DNA clean-up step with AMPure® beads. with a limit of detection of 6 eggs of T. canis spiked in 10 g of soil with a probability of 97%. Conclusion The pre-analytical flow process developed here combined with qPCR represents an improved method for the surveillance of Toxocara contamination in the environment. Parasitology Toxocara canis Toxocara cati helminth eggs soil sand inhibitors DNA extraction qPCR clean-up Figures Figure 1 Figure 2 Background T. canis and T. cati are parasitic roundworms that are causative agents of toxocarosis, a widespread neglected zoonotic disease [ 1 , 2 , 3 ], that afflicts millions of people worldwide [ 4 ]. The disease is transmitted to humans through ingestion of contaminated soil containing T. canis eggs from faeces of dogs and T. cati from faeces of cats [ 5 ]. The understanding of the extent of environmental contamination with Toxocara spp. eggs, including the relative importance of different definitive hosts as sources of ova is listed as one of the knowledge gaps in the epidemiology of Toxocara [ 2 ]. Despite the development of sensitive quantitative PCR (qPCR) assays for detection T. canis and T. cati eggs [ 6 ], low abundance of Toxocara eggs in environmental samples still constitutes a shortcoming in the control of toxocarosis [ 7 ]. This shortcoming is compounded by the low effectiveness of DNA extraction methods available for Toxocara eggs in soil samples. Whereas several protocols for obtaining helminth DNA from environmental samples have been developed [ 8 ], they have not displayed an efficient effectiveness for Toxocara eggs in soil samples [ 9 ]. A method combining Toxocara eggs enrichment by the flotation technique and subsequent DNA extraction from soil samples spiked with Toxocara eggs has resulted in the detection rate of Toxocara in 41.7% of 10 g of soil samples spiked with 10 eggs, and in only 8.3% for samples spiked with 1 egg [ 9 ]. These results underscore the need for alternative methods for sensitive detection of Toxocara eggs in soil samples. The aim of the study was to optimize DNA extraction of T. canis eggs from soil and sand samples. This entails: i) efficient Toxocara eggs disruption, ii) efficient DNA extraction and iii) removal of PCR inhibitors which might be present in DNA solutions. This is expected to improve PCR-based detection of Toxocara in environmental samples. Methods Soil samples collections Soil samples (250 g) were collected according to systematic unaligned sampling method, from the upper soil layer of 3 cm [ 10 ]. The samples were then dried for 24–48 h and sifted through a 2-mm sieve to remove stones and larger organic particles. Egg-spiking experiments were carried out in presumably Toxocara -free clean sand and soil samples (i.e. commercial sand and soil from a backyard without any history of dog, cat or fox presence). Likewise, 250 g of soil samples (n = 40) were collected in and around Tuliszków (Poland, 52°04′35″N – 18°17′37″E), covering also nearby rural villages. The sites included playgrounds (n = 20) and backyards close to households (n = 20). Egg stock solution and serial dilutions T. canis fertilized eggs were isolated from uteri of adult female worms and suspended in DNA/RNA-free water. 4 aliquots of 5 µL of the unembryonated eggs suspensions were observed under the light microscope (100-fold magnification). The number of eggs was calculated per field and then reported as the grand mean of the 8 squares ± standard deviation (SD). Serial dilutions (i.e., 10 4 , 10 3 , 10 2 , 10, 1) eggs were prepared in DNA/RNA-free water. Eggs shell disruption methods Six methods for disruption of T. canis eggs (1, 10, 10 2 and 10 3 eggs suspensions) were compared: 1) enzymatic lysis with proteinase K (PK) (incubation of eggs solution with 0.2 unit of proteinase K in 40 µL solution containing 10% (w/v) of SDS at 56°C under agitation at 800 rpm during 2 hours); 2) thermal disruption (TL) (5 freeze-thaw cycles: 3 min of freezing in liquid nitrogen, followed by 3 min of thaw in boiling water under agitation at 800 rpm); 3) mechanical disruption of eggs using FastPrep® tubes containing the lysing matrix A beads (FPA) (MP Biomedicals, Santa Ana, California, USA) under shaking at 6m/s during 40 seconds in a FastPrep-24 homogenizer (3 cycles); 4) the same protocol as the previous but using lysing matrix D beads (FPD) instead; 5) TL followed by FPD (TL-FPD); and 6) TL-FPD followed by PK (TL-FPD-PK). Following the disruption step, DNA was extracted using the Nuclisens® MiniMag® Kit (BioMérieux, Boxtel, The Netherlands) according to the manufacturer’s protocol. DNA solutions were kept at -20°C use. DNA extraction with commercial kits from sand and soil samples Toxocara -free soil and sand samples (10g) were spiked in triplicate with 1, 10, 10 2 , 10 3 and 10 4 eggs of T. canis. For the selection of the more efficient DNA extraction method in sand and soil samples, two kits comprising the best disruption method were compared on sand and soil samples spiked with 10 4 eggs. Direct DNA extraction was performed on the samples spiked with 10 4 eggs using the DNeasy® PowerMax® Soil Kit (Qiagen, Hilden, Germany) and the FastDNA™ SPIN Kit for Soil (MP Biomedicals, Santa Ana, California, USA) according to the manufacturer instructions. The extraction method associated with a lower limit of detection for Toxocara in soil samples (10 g of soil spiked 10 4 eggs) was selected for further detection of Toxocara eggs in soil samples spiked with 10 3 , 10 2 , 10, 1 eggs of T. canis eggs. Likewise, the selected method was used for DNA extraction from soil samples (n = 40) collected in the rural area around Tuliszków in Poland for detection of T. canis/T. cati eggs using the duplex qPCR developped previously (Durant et al, 2012). DNA clean-up The impact of a clean-up step for removal of PCR inhibitors was assessed by performing duplex qPCR for T. canis and T. cati on DNA before and after a clean-up step. A magnetic beads DNA method (Agencourt® AMPure®, Beckman Coulter, Massachusetts, USA) was used for DNA clean-up (i.e. for removal of PCR inhibitors) in all samples. Briefly, 1.8 volume of AMPure® beads was added to 1 volume of extracted DNA. Bind DNA was placed on a magnetic stand and the solution was discarded. The DNA was then washed 2x with 70% ethanol. DNA was eluted in 1 volume of DNA/RNA free water. All purified DNA solutions were kept at -20°C until use. Quantitative real-time PCR Specific quantitative real-time polymerase chain reaction (qPCR) targeting T. canis (for samples spiked with eggs) or T. canis and T. cati (for environmental samples) was carried out in triplicate on extracted DNA according to the procedure previously described by Durant et al. (2012) on a CFX-96 thermocycler (Bio-Rad, Hercules, California, USA). An Ascaridoidea-generic qPCR was used as an internal quality control [ 6 ]. Flotation method and microscopic observation Environmental samples were processed using the flotation-centrifugation method [ 11 ] using the sugar Sheather’s solution of specific gravity 1.27. Two coverslips were used to recover eggs from each sample, examined under light microscopy for counting of T. canis eggs. Recovered eggs were characterized based on their size, the thickness of eggshells, transparency and visibility of semi-circular cavities on their surfaces. Statistical analysis For each of the egg disruption methods tested, extraction yield was calculated on the base of the qPCR Cq values. When only 1 PCR reaction from the 3 replicates led to a missing value, this missing value was excluded from downstream analysis, as described and recommended in a recent survey on qPCR data analysis [ 12 ]. A calibration curve was built for each method and the Limit of Detection (LoD) [ 13 ] was calculated at the intersection between the Limit of Blank (LoB) and the prediction interval of the calibration curve. In the current study, the LoD is therefore the lowest number of eggs likely to be reliably distinguished from the LoB and at which detection is feasible. To determine the concordance between results obtained with tested extraction methods, the Kappa statistics of Cohen was calculated. All statistical analyses were performed using R.3.4 and the SSPS® Statistics software (IBM, New York, USA). Results Dilution series of eggs disruption methods The comparison of T. canis eggs disruption methods is shown in Fig. 1. TL, FPD and TL-FPD-PK (LoD = 7 eggs) were the three disruption methods displaying the lowest LoD with the best yield. FPD was selected over TL-FPD-PK as it is handy and easy to use in the field. Accordingly, DNA extraction kits which include a mechanical eggs disruption step were selected for assessment of the presence of T. canis in sand and soil samples spiked by serial dilutions of T. canis eggs. Practically, the DNeasy® PowerMax® Soil Kit (Qiagen, Hilden, Germany) and the FastDNA™ SPIN Kit for Soil (MP Biomedicals, Santa Ana, California, USA) were used for DNA extraction according to the manufacturer instructions. DNA extraction with commercial kits from sand and soil samples Cq values from sand and soil samples spiked with 10 4 eggs of T. canis are shown in Table 1 . Table 1 Comparison of DNA extraction efficiency on 10 4 eggs spiked in soil and sand samples (10 g) when using two specific kits. Commercial kits Types of samples Before clean-up After AMPure® clean-up 1:1 1:10 dilution 1:1 1:10 dilution DNeasy® PowerMax® Soil Kit Sand 24.32 ± 0.60 26.61 ± 0.22 24.02 ± 0.14 27.45 ± 0.09 Soil Negative Negative 29.23 ± 1.32 31.32 ± 0.31 FastDNA™ SPIN Kit for Soil Sand 34.69 ± 0.23 37.66 ± 0.24 37.14 ± 1.11 Negative Soil Negative Negative Negative 30.03 ± 1.00 The extraction efficiency is reflected through Cq values of qPCR In spiked sand samples, DNeasy® PowerMax® Soil Kit displayed lower Cq values in comparison with the FastDNA™ SPIN Kit for Soil. However, whereas detection of T. canis in sand samples spiked with 10 4 eggs was straightforward, this was not the case for soil samples. Indeed, no positive qPCR signal was recorded in soil samples spiked with the same amount of T. canis eggs prior to the clean-up step. The clean-up step improved the detection of T. canis in soil samples, suggesting that this step was critical for the removal of PCR inhibitors in these samples. Overall, DNeasy® PowerMax® Soil Kit associated with AMPure® clean-up improved the detection However, by looking closely at delta Cq values between non-diluted DNA and 1:10 diluted DNA for sand and soil samples respectively, (3.43 vs 2.09) the low delta Cq values in soil samples suggest that despite the improvement by AMPure® clean-up, not all inhibitors were removed in soil samples. Notwithstanding these limitations, the DNeasy® PowerMax® Soil Kit plus a clean-up step with AMPure® beads was selected as the optimal method for DNA extraction in soil samples. Indeed, the limit of detection on DNA solutions extracted from soil samples spiked with 10 3 , 10 2 , 10, 1 eggs of T. canis using the DNeasy® PowerMax® Soil Kit combined with clean-up step with AMPure® beads was 6 eggs in 10 g of soil with a probability of 97% (Fig. 2). Accordingly, this method was used for the detection of T. canis/T. cati eggs in soil samples (n = 40) collected in the rural area around Tuliszków in Poland. Environmental samples analyses A total of 40 environmental samples were assessed for the presence of Toxocara eggs through the processing of 10 g of soil (from a total of 250 g) using the protocol described above. In parallel, 40 g soil samples were processed through the conventional microscopic examination after an enrichment (flotation) step. qPCR and microscopic observation results are summarized in Table 2 . Table 2 Cq values (mean ± SD) and light microscopic observation results on 40 environmental samples Sample N° Type qPCR Microscopic observation 1 Playground Negative Negative 2 Playground Negative Negative 3 Playground 34.33 ± 0.51 Negative 4 Playground Negative Negative 5 Playground Negative Negative 6 Playground Negative Negative 7 Playground 35.99 ± 0.68 Negative 8 Playground 33.41 ± 0.55 Negative 9 Playground Negative Negative 10 Playground Negative Negative 11 Playground Negative Negative 12 Playground Negative Negative 13 Playground Negative Negative 14 Playground Negative 1 T. canis egg with a larva 15 Playground Negative Negative 16 Playground Negative Negative 17 Playground 33.73 ± 1.04 Negative 18 Playground Negative Negative 19 Playground Negative Negative 20 Playground Negative Negative 21 Backyard Negative Negative 22 Backyard Negative Negative 23 Backyard Negative Negative 24 Backyard Negative Negative 25 Backyard Negative Negative 26 Backyard Negative Negative 27 Backyard Negative Negative 28 Backyard Negative Negative 29 Backyard Negative Negative 30 Backyard Negative Negative 31 Backyard Negative Negative 32 Backyard 24.30 ± 0.09 7 T. canis eggs with a larva 33 Backyard 30.55 ± 0.23 Negative 34 Backyard Negative Negative 35 Backyard Negative Negative 36 Backyard 29.72 ± 0.04 1 T. canis egg 37 Backyard 23.30 ± 0.31 12 T. canis eggs with a larva 38 Backyard Negative Negative 39 Backyard Negative Negative 40 Backyard Negative Negative Samples 7 and 14 come from the same playground. Likewise, samples 31, 32 et 40 are coming from the same backyard. Altogether, the duplex-specific qPCR was positive for T. canis in 8/40 samples while no signal relating to the presence of T. cati was generated and the Ascaridoidea - generic qPCR was consistently positive in all samples outlining the presence of Ascaridoidea DNA in all environmental samples examined. Overall, there was a moderate agreement between light microscopic observation on enriched soil samples after flotation, with Cohen κ = 0.423 ( p < 0.005). However, the molecular assay (i.e, DNA extraction using the DNeasy® PowerMax® Soil Kit plus AMPure® clean-up and duplex qPCR) allowed the detection of more positive samples when compared to light microscopy observation on flotation-enriched samples. Discussion Soil is considered the primary source of Toxocara transmission to humans, especially children [ 14 ]. Soil contamination by eggs of Toxocara spp. is due to the presence of dogs and cats, two companion animals which are widely distributed in the world. This wide distribution of hosts is compounded by the features of Toxocara eggs, which can survive in the environment for months or even years [ 15 ]. It is therefore crucial to establish environmental surveillance programs aimed at detecting Toxocara eggs in soil, especially in places often crossed by dogs and cats and which happen to be frequented by children. However, detection of Toxocara eggs in soil samples still faces serious hurdles, which are in most parts due to the lack of a reliable sensitive method aimed at this objective. So far, enrichment by flotation and subsequent light microscopic examination remains the most used method for surveillance of soil contamination with Toxocara eggs. Needless to recall, this method is difficult to perform in the field, is time consuming and requires experience for light microscopy and parasitological diagnostics. By using a DNA extraction method including a bead-beating step for disruption of the thick wall of Toxocara eggs and a clean-up step for removal of PCR inhibitors, we have managed to achieve a sensitivity of 6 eggs in 10 g of soil. To the best of our knowledge, this is an improvement compared to previously published methods [ 6 , 9 ]. A method combining flotation and qPCR achieved a detection threshold of 10 eggs for the flatworm Echinococcus multilocularis per 10 g of soil but failed to reach a similar figure for Toxocara eggs [ 9 ]. Whereas the causes behind this low sensitivity for detection of Toxocara are not known, they might be associated with the use of methods which did not put emphasis on the mandatory disruption of Toxocara eggs prior to DNA extraction. By improving this step, we have been able to achieve a sensitivity level of Toxocara comparable to that observed with other roundworms. However, as shown by the results of spiking of soil, the total removal of PCR inhibitors cannot be guaranteed, as pointed out by other authors [ 16 ]. Another hurdle in the detection of control of soil contamination with Toxocara eggs is the non-homogenous distribution of Toxocara eggs in soil samples, even those sampled from the same backyards or playgrounds. As an illustration, samples 7 and 14 which came from the same playground did not display similar qPCR results. A solution might reside in sampling more areas. Also, PCR inhibitors as the cause of a PCR negative result cannot totally be ruled out. The same observation applies to samples 31, 32 and 40 which came from the same backyard. Of note, the alleged improved sensitivity on detection of helminth such as Echinococcus multilocularis was achieved by combining an enrichment (flotation) step with qPCR [ 9 ]. This renders the method cumbersome and time-consuming, two features which preclude the use of the method in the field for screening of hundreds of environmental samples. There are several clean-up kits for removal of PCR inhibitors. In our assay, The AMPure® beads purification was compared with the PowerClean Pro Cleanup Kit (Qiagen, Hilden, Germany) and displayed similar efficiency with respect to removal of PCR inhibitors (data not shown). The AMPure® clean-up was favoured as it is compatible with the prospect of automation of the whole analytical processing. One lingering question is whether positive results from qPCR systematically can be taken at value face as a proof of soil contamination, given that dead eggs of Toxocara can be associated with positive qPCR signals without also being able to cause toxocarosis when ingested by humans. Whereas no definitive answer for this question is available, the fact that Toxocara eggs can survive in the environment for several years [ 15 ] and the probability that viable eggs can be mistaken for dead eggs upon light microscopic observation should prompt us to consider qPCR positive as a reliable marker of soil contamination with Toxocara eggs. Conclusions In conclusion, the processing developed here and which combines a mechanical disruption of Toxocara eggs, DNA extraction using the DNeasy® PowerMax® Soil Kit and a subsequent DNA clean-up for removal of PCR inhibitors improves substantially detection of Toxocara eggs in soil samples by qPCR. Moreover, the potential for automation offered by this method can result in processing of hundreds of soil samples and allow to deliver timely results in a short period. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Funding This work was supported by the Wallonie-Bruxelles international agency (project 320167) with the contribution of the Dean of Faculty of Health Sciences, Poznań University of Physical Education. Authors’ contributions JW, DJF, MWH and GJL conceived the study; JW, FWR and MWH provided eggs suspensions and environmental samples; JW carried out microscopic examination; DJF and WJ carried out molecular analyses; JW and DJF wrote the first draft of the paper, and JW, DJF, ILM, and JLG contributed to the final manuscript which they approve. Acknowledgments We gratefully acknowledge Tomasz Luty (DVM), PhD, for delivering Toxocara canis worms and Jérôme Ambroise (CTMA), PhD, for his statistical analysis contributions. References Chen J, Zhou DH, Nisbet AJ, Xu MJ, Huang SY, Li MW, et al. Advances in molecular identification, taxonomy, genetic variation and diagnosis of Toxocara spp. Infect Genet Evol. 2012;12 7:1344–8; doi: 10.1016/j.meegid.2012.04.019 . https://www.ncbi.nlm.nih.gov/pubmed/22569289 . Holland CV. Knowledge gaps in the epidemiology of Toxocara: the enigma remains. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-264679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":13364563,"identity":"0e669fcb-9a9d-46fd-a756-31203be513f3","order_by":0,"name":"Wojciech Jarosz","email":"","orcid":"","institution":"Poznan University of Physical Education","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Jarosz","suffix":""},{"id":13364564,"identity":"9409070c-32a5-4c6e-8c18-6b9ff356ce31","order_by":1,"name":"Jean-François Durant","email":"","orcid":"","institution":"Université catholique de Louvain: Universite Catholique de Louvain","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jean-François","middleName":"","lastName":"Durant","suffix":""},{"id":13364565,"identity":"b76f79db-bfa4-42e8-b025-6b1fc6ffec0e","order_by":2,"name":"Leonid Mwana wa bene Irenge","email":"","orcid":"","institution":"Université catholique de Louvain: Universite Catholique de Louvain","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Leonid","middleName":"Mwana wa bene","lastName":"Irenge","suffix":""},{"id":13364566,"identity":"d9f8761c-42e7-4d1e-a792-67dbd4445442","order_by":3,"name":"Renata Fogt-Wyrwas","email":"","orcid":"","institution":"Akademia Wychowania Fizycznego im Eugeniusza Piaseckiego w Poznaniu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Renata","middleName":"","lastName":"Fogt-Wyrwas","suffix":""},{"id":13364567,"identity":"a993987b-ac6f-4cb7-af49-773b14207b3b","order_by":4,"name":"Hanna Mizgajska-Wiktor","email":"","orcid":"","institution":"Poznan University of Physical Education","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanna","middleName":"","lastName":"Mizgajska-Wiktor","suffix":""},{"id":13364568,"identity":"563a61b9-429f-4346-9dbd-bab48a2b9c0e","order_by":5,"name":"Jean-Luc Gala","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-7749-573X","institution":"Université catholique de Louvain: Universite Catholique de Louvain","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jean-Luc","middleName":"","lastName":"Gala","suffix":""}],"badges":[],"createdAt":"2021-02-21 19:43:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-264679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-264679/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6369240,"identity":"8ac184c0-1aa5-4c20-8439-c6e73a78b4f8","added_by":"auto","created_at":"2021-02-25 21:57:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27142,"visible":true,"origin":"","legend":"Comparison of eggs disruption methods. Disruption methods are reflected by Cq values \nLimit of Detection (LoD) of 6 eggs disruption methods. The different methods are compared with non-disrupted eggs.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-264679/v1/acbb3ead5f2277ff94634657.png"},{"id":6369377,"identity":"dbe47f92-86a8-4f22-9264-11f25561dd25","added_by":"auto","created_at":"2021-02-25 22:00:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18112,"visible":true,"origin":"","legend":"LoD for T. canis eggs in spiked soil samples.\nCq values (y-axis) are plotted against the number of eggs (x-axis). The LoD is expressed as the number of eggs detected with a probability of 97%. \n","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-264679/v1/abf46b30267e1c993b25926c.png"},{"id":15671057,"identity":"723ef61d-bc33-4ab5-bdaf-162dfb03c2c2","added_by":"auto","created_at":"2021-11-18 14:04:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":512812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-264679/v1/38ed1ca2-187f-4154-aabc-aec447cd86d8.pdf"},{"id":6369378,"identity":"078c8243-2eef-4150-8a53-e0283e9ba1a0","added_by":"auto","created_at":"2021-02-25 22:00:41","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":113828,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-264679/v1/d31e72b323a01650609c3271.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eOptimized DNA-based Identification of \u003cem\u003eToxocara \u003c/em\u003espp. Egg in Difficult Matrices: A Case for Specific and Sensitive Identification of Geohelminth Eggs\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003e \u003cem\u003eT. canis\u003c/em\u003e and \u003cem\u003eT. cati\u003c/em\u003e are parasitic roundworms that are causative agents of toxocarosis, a widespread neglected zoonotic disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], that afflicts millions of people worldwide [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The disease is transmitted to humans through ingestion of contaminated soil containing \u003cem\u003eT. canis\u003c/em\u003e eggs from faeces of dogs and \u003cem\u003eT. cati\u003c/em\u003e from faeces of cats [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The understanding of the extent of environmental contamination with \u003cem\u003eToxocara\u003c/em\u003e spp. eggs, including the relative importance of different definitive hosts as sources of ova is listed as one of the knowledge gaps in the epidemiology of \u003cem\u003eToxocara\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the development of sensitive quantitative PCR (qPCR) assays for detection \u003cem\u003eT. canis\u003c/em\u003e and \u003cem\u003eT. cati\u003c/em\u003e eggs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], low abundance of \u003cem\u003eToxocara\u003c/em\u003e eggs in environmental samples still constitutes a shortcoming in the control of toxocarosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This shortcoming is compounded by the low effectiveness of DNA extraction methods available for \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples. Whereas several protocols for obtaining helminth DNA from environmental samples have been developed [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], they have not displayed an efficient effectiveness for \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A method combining \u003cem\u003eToxocara\u003c/em\u003e eggs enrichment by the flotation technique and subsequent DNA extraction from soil samples spiked with \u003cem\u003eToxocara\u003c/em\u003e eggs has resulted in the detection rate of \u003cem\u003eToxocara\u003c/em\u003e in 41.7% of 10 g of soil samples spiked with 10 eggs, and in only 8.3% for samples spiked with 1 egg [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These results underscore the need for alternative methods for sensitive detection of \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples. The aim of the study was to optimize DNA extraction of \u003cem\u003eT. canis\u003c/em\u003e eggs from soil and sand samples. This entails: i) efficient \u003cem\u003eToxocara\u003c/em\u003e eggs disruption, ii) efficient DNA extraction and iii) removal of PCR inhibitors which might be present in DNA solutions. This is expected to improve PCR-based detection of \u003cem\u003eToxocara\u003c/em\u003e in environmental samples.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eSoil samples collections\u003c/h2\u003e\n\u003cp\u003eSoil samples (250 g) were collected according to systematic unaligned sampling method, from the upper soil layer of 3 cm [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. The samples were then dried for 24\u0026ndash;48 h and sifted through a 2-mm sieve to remove stones and larger organic particles. Egg-spiking experiments were carried out in presumably \u003cem\u003eToxocara\u003c/em\u003e-free clean sand and soil samples (i.e. commercial sand and soil from a backyard without any history of dog, cat or fox presence). Likewise, 250 g of soil samples (n\u0026thinsp;=\u0026thinsp;40) were collected in and around Tuliszk\u0026oacute;w (Poland, 52\u0026deg;04\u0026prime;35\u0026Prime;N \u0026ndash; 18\u0026deg;17\u0026prime;37\u0026Prime;E), covering also nearby rural villages. The sites included playgrounds (n\u0026thinsp;=\u0026thinsp;20) and backyards close to households (n\u0026thinsp;=\u0026thinsp;20).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eEgg stock solution and serial dilutions\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eT. canis\u003c/em\u003e fertilized eggs were isolated from uteri of adult female worms and suspended in DNA/RNA-free water. 4 aliquots of 5 \u0026micro;L of the unembryonated eggs suspensions were observed under the light microscope (100-fold magnification). The number of eggs was calculated per field and then reported as the grand mean of the 8 squares \u0026plusmn; standard deviation (SD). Serial dilutions (i.e., 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, 10, 1) eggs were prepared in DNA/RNA-free water.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eEggs shell disruption methods\u003c/h2\u003e\n\u003cp\u003eSix methods for disruption of \u003cem\u003eT. canis\u003c/em\u003e eggs (1, 10, 10\u003csup\u003e2\u003c/sup\u003e and 10\u003csup\u003e3\u003c/sup\u003e eggs suspensions) were compared: 1) enzymatic lysis with proteinase K (PK) (incubation of eggs solution with 0.2 unit of proteinase K in 40 \u0026micro;L solution containing 10% (w/v) of SDS at 56\u0026deg;C under agitation at 800 rpm during 2 hours); 2) thermal disruption (TL) (5 freeze-thaw cycles: 3 min of freezing in liquid nitrogen, followed by 3 min of thaw in boiling water under agitation at 800 rpm); 3) mechanical disruption of eggs using FastPrep\u0026reg; tubes containing the lysing matrix A beads (FPA) (MP Biomedicals, Santa Ana, California, USA) under shaking at 6m/s during 40 seconds in a FastPrep-24 homogenizer (3 cycles); 4) the same protocol as the previous but using lysing matrix D beads (FPD) instead; 5) TL followed by FPD (TL-FPD); and 6) TL-FPD followed by PK (TL-FPD-PK). Following the disruption step, DNA was extracted using the Nuclisens\u0026reg; MiniMag\u0026reg; Kit (BioM\u0026eacute;rieux, Boxtel, The Netherlands) according to the manufacturer\u0026rsquo;s protocol. DNA solutions were kept at -20\u0026deg;C use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eDNA extraction with commercial kits from sand and soil samples\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eToxocara\u003c/em\u003e-free soil and sand samples (10g) were spiked in triplicate with 1, 10, 10\u003csup\u003e2\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e4\u003c/sup\u003e eggs of \u003cem\u003eT. canis.\u003c/em\u003e For the selection of the more efficient DNA extraction method in sand and soil samples, two kits comprising the best disruption method were compared on sand and soil samples spiked with 10\u003csup\u003e4\u003c/sup\u003e eggs. Direct DNA extraction was performed on the samples spiked with 10\u003csup\u003e4\u003c/sup\u003e eggs using the DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit (Qiagen, Hilden, Germany) and the FastDNA\u0026trade; SPIN Kit for Soil (MP Biomedicals, Santa Ana, California, USA) according to the manufacturer instructions. The extraction method associated with a lower limit of detection for \u003cem\u003eToxocara\u003c/em\u003e in soil samples (10 g of soil spiked 10\u003csup\u003e4\u003c/sup\u003e eggs) was selected for further detection of \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples spiked with 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, 10, 1 eggs of \u003cem\u003eT. canis\u003c/em\u003e eggs. Likewise, the selected method was used for DNA extraction from soil samples (n\u0026thinsp;=\u0026thinsp;40) collected in the rural area around Tuliszk\u0026oacute;w in Poland for detection of \u003cem\u003eT. canis/T. cati\u003c/em\u003e eggs using the duplex qPCR developped previously (Durant et al, 2012).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eDNA clean-up\u003c/h2\u003e\n\u003cp\u003eThe impact of a clean-up step for removal of PCR inhibitors was assessed by performing duplex qPCR for \u003cem\u003eT. canis\u003c/em\u003e and \u003cem\u003eT. cati\u003c/em\u003e on DNA before and after a clean-up step. A magnetic beads DNA method (Agencourt\u0026reg; AMPure\u0026reg;, Beckman Coulter, Massachusetts, USA) was used for DNA clean-up (i.e. for removal of PCR inhibitors) in all samples. Briefly, 1.8 volume of AMPure\u0026reg; beads was added to 1 volume of extracted DNA. Bind DNA was placed on a magnetic stand and the solution was discarded. The DNA was then washed 2x with 70% ethanol. DNA was eluted in 1 volume of DNA/RNA free water. All purified DNA solutions were kept at -20\u0026deg;C until use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e\n\u003cp\u003eSpecific quantitative real-time polymerase chain reaction (qPCR) targeting \u003cem\u003eT. canis\u003c/em\u003e (for samples spiked with eggs) or \u003cem\u003eT. canis\u003c/em\u003e and \u003cem\u003eT. cati\u003c/em\u003e (for environmental samples) was carried out in triplicate on extracted DNA according to the procedure previously described by Durant et al. (2012) on a CFX-96 thermocycler (Bio-Rad, Hercules, California, USA). An Ascaridoidea-generic qPCR was used as an internal quality control [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eFlotation method and microscopic observation\u003c/h2\u003e\n\u003cp\u003eEnvironmental samples were processed using the flotation-centrifugation method [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e] using the sugar Sheather\u0026rsquo;s solution of specific gravity 1.27. Two coverslips were used to recover eggs from each sample, examined under light microscopy for counting of \u003cem\u003eT. canis\u003c/em\u003e eggs. Recovered eggs were characterized based on their size, the thickness of eggshells, transparency and visibility of semi-circular cavities on their surfaces.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eFor each of the egg disruption methods tested, extraction yield was calculated on the base of the qPCR Cq values. When only 1 PCR reaction from the 3 replicates led to a missing value, this missing value was excluded from downstream analysis, as described and recommended in a recent survey on qPCR data analysis [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. A calibration curve was built for each method and the Limit of Detection (LoD) [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] was calculated at the intersection between the Limit of Blank (LoB) and the prediction interval of the calibration curve. In the current study, the LoD is therefore the lowest number of eggs likely to be reliably distinguished from the LoB and at which detection is feasible. To determine the concordance between results obtained with tested extraction methods, the Kappa statistics of Cohen was calculated. All statistical analyses were performed using R.3.4 and the SSPS\u0026reg; Statistics software (IBM, New York, USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eDilution series of eggs disruption methods\u003c/h2\u003e\n\u003cp\u003eThe comparison of \u003cem\u003eT. canis\u003c/em\u003e eggs disruption methods is shown in Fig.\u0026nbsp;1. TL, FPD and TL-FPD-PK (LoD\u0026thinsp;=\u0026thinsp;7 eggs) were the three disruption methods displaying the lowest LoD with the best yield. FPD was selected over TL-FPD-PK as it is handy and easy to use in the field. Accordingly, DNA extraction kits which include a mechanical eggs disruption step were selected for assessment of the presence of \u003cem\u003eT. canis\u003c/em\u003e in sand and soil samples spiked by serial dilutions of \u003cem\u003eT. canis\u003c/em\u003e eggs. Practically, the DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit (Qiagen, Hilden, Germany) and the FastDNA\u0026trade; SPIN Kit for Soil (MP Biomedicals, Santa Ana, California, USA) were used for DNA extraction according to the manufacturer instructions.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eDNA extraction with commercial kits from sand and soil samples\u003c/h2\u003e\n\u003cp\u003eCq values from sand and soil samples spiked with 10\u003csup\u003e4\u003c/sup\u003e eggs of \u003cem\u003eT. canis\u003c/em\u003e are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of DNA extraction efficiency on 10\u003csup\u003e4\u003c/sup\u003e eggs spiked in soil and sand samples (10 g) when using two specific kits.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCommercial kits\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTypes of\u003c/p\u003e\n\u003cp\u003esamples\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eBefore clean-up\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eAfter AMPure\u0026reg; clean-up\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1:1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1:10 dilution\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1:1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1:10 dilution\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDNeasy\u0026reg; PowerMax\u0026reg; Soil Kit\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSand\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e24.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e26.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e24.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e27.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSoil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e29.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e31.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 70px;\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFastDNA\u0026trade; SPIN Kit for Soil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSand\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e34.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e37.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e37.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSoil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e30.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003cp\u003eThe extraction efficiency is reflected through Cq values of qPCR\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn spiked sand samples, DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit displayed lower Cq values in comparison with the FastDNA\u0026trade; SPIN Kit for Soil. However, whereas detection of\u0026nbsp;\u003cem\u003eT. canis\u003c/em\u003e\u0026nbsp;in sand samples spiked with 10\u003csup\u003e4\u003c/sup\u003e\u0026nbsp;eggs was straightforward, this was not the case for soil samples. Indeed, no positive qPCR signal was recorded in soil samples spiked with the same amount of\u0026nbsp;\u003cem\u003eT. canis\u003c/em\u003e\u0026nbsp;eggs prior to the clean-up step. The clean-up step improved the detection of\u0026nbsp;\u003cem\u003eT. canis\u003c/em\u003e\u0026nbsp;in soil samples, suggesting that this step was critical for the removal of PCR inhibitors in these samples. Overall, DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit associated with AMPure\u0026reg; clean-up improved the detection However, by looking closely at delta Cq values between non-diluted DNA and 1:10 diluted DNA for sand and soil samples respectively, (3.43 vs 2.09) the low delta Cq values in soil samples suggest that despite the improvement by AMPure\u0026reg; clean-up, not all inhibitors were removed in soil samples. Notwithstanding these limitations, the DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit plus a clean-up step with AMPure\u0026reg; beads was selected as the optimal method for DNA extraction in soil samples. Indeed, the limit of detection on DNA solutions extracted from soil samples spiked with 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, 10, 1 eggs of\u0026nbsp;\u003cem\u003eT. canis\u003c/em\u003e\u0026nbsp;using the DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit combined with clean-up step with AMPure\u0026reg; beads was 6 eggs in 10 g of soil with a probability of 97% (Fig.\u0026nbsp;2). Accordingly, this method was used for the detection of\u0026nbsp;\u003cem\u003eT. canis/T. cati\u003c/em\u003e\u0026nbsp;eggs in soil samples (n\u0026thinsp;=\u0026thinsp;40) collected in the rural area around Tuliszk\u0026oacute;w in Poland.\u003c/p\u003e\n\u003ch2\u003eEnvironmental samples analyses\u003c/h2\u003e\n\u003cp\u003eA total of 40 environmental samples were assessed for the presence of \u003cem\u003eToxocara\u003c/em\u003e eggs through the processing of 10 g of soil (from a total of 250 g) using the protocol described above. In parallel, 40 g soil samples were processed through the conventional microscopic examination after an enrichment (flotation) step. qPCR and microscopic observation results are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCq values (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) and light microscopic observation results on 40 environmental samples\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSample N\u0026deg;\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eType\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eqPCR\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMicroscopic observation\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 \u003cem\u003eT. canis\u003c/em\u003e egg with a larva\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlayground\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 \u003cem\u003eT. canis\u003c/em\u003e eggs with a larva\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 \u003cem\u003eT. canis\u003c/em\u003e egg\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12 \u003cem\u003eT. canis\u003c/em\u003e eggs with a larva\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBackyard\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eSamples 7 and 14 come from the same playground. Likewise, samples 31, 32 et 40 are coming from the same backyard.\u003c/p\u003e\n\u003cp\u003eAltogether, the duplex-specific qPCR was positive for \u003cem\u003eT. canis\u003c/em\u003e in 8/40 samples while no signal relating to the presence of \u003cem\u003eT. cati\u003c/em\u003e was generated and the Ascaridoidea\u003cem\u003e-\u003c/em\u003egeneric qPCR was consistently positive in all samples outlining the presence of Ascaridoidea DNA in all environmental samples examined.\u003c/p\u003e\n\u003cp\u003eOverall, there was a moderate agreement between light microscopic observation on enriched soil samples after flotation, with Cohen \u0026kappa;\u0026thinsp;=\u0026thinsp;0.423 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005). However, the molecular assay (i.e, DNA extraction using the DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit plus AMPure\u0026reg; clean-up and duplex qPCR) allowed the detection of more positive samples when compared to light microscopy observation on flotation-enriched samples.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eSoil is considered the primary source of \u003cem\u003eToxocara\u003c/em\u003e transmission to humans, especially children [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Soil contamination by eggs of \u003cem\u003eToxocara\u003c/em\u003e spp. is due to the presence of dogs and cats, two companion animals which are widely distributed in the world. This wide distribution of hosts is compounded by the features of \u003cem\u003eToxocara\u003c/em\u003e eggs, which can survive in the environment for months or even years [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is therefore crucial to establish environmental surveillance programs aimed at detecting \u003cem\u003eToxocara\u003c/em\u003e eggs in soil, especially in places often crossed by dogs and cats and which happen to be frequented by children. However, detection of \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples still faces serious hurdles, which are in most parts due to the lack of a reliable sensitive method aimed at this objective. So far, enrichment by flotation and subsequent light microscopic examination remains the most used method for surveillance of soil contamination with \u003cem\u003eToxocara\u003c/em\u003e eggs. Needless to recall, this method is difficult to perform in the field, is time consuming and requires experience for light microscopy and parasitological diagnostics.\u003c/p\u003e \u003cp\u003eBy using a DNA extraction method including a bead-beating step for disruption of the thick wall of \u003cem\u003eToxocara\u003c/em\u003e eggs and a clean-up step for removal of PCR inhibitors, we have managed to achieve a sensitivity of 6 eggs in 10 g of soil. To the best of our knowledge, this is an improvement compared to previously published methods [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA method combining flotation and qPCR achieved a detection threshold of 10 eggs for the flatworm \u003cem\u003eEchinococcus multilocularis\u003c/em\u003e per 10 g of soil but failed to reach a similar figure for \u003cem\u003eToxocara\u003c/em\u003e eggs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Whereas the causes behind this low sensitivity for detection of \u003cem\u003eToxocara\u003c/em\u003e are not known, they might be associated with the use of methods which did not put emphasis on the mandatory disruption of \u003cem\u003eToxocara\u003c/em\u003e eggs prior to DNA extraction. By improving this step, we have been able to achieve a sensitivity level of \u003cem\u003eToxocara\u003c/em\u003e comparable to that observed with other roundworms. However, as shown by the results of spiking of soil, the total removal of PCR inhibitors cannot be guaranteed, as pointed out by other authors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother hurdle in the detection of control of soil contamination with \u003cem\u003eToxocara\u003c/em\u003e eggs is the non-homogenous distribution of \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples, even those sampled from the same backyards or playgrounds. As an illustration, samples 7 and 14 which came from the same playground did not display similar qPCR results. A solution might reside in sampling more areas. Also, PCR inhibitors as the cause of a PCR negative result cannot totally be ruled out. The same observation applies to samples 31, 32 and 40 which came from the same backyard. Of note, the alleged improved sensitivity on detection of helminth such as \u003cem\u003eEchinococcus multilocularis\u003c/em\u003e was achieved by combining an enrichment (flotation) step with qPCR [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This renders the method cumbersome and time-consuming, two features which preclude the use of the method in the field for screening of hundreds of environmental samples.\u003c/p\u003e \u003cp\u003eThere are several clean-up kits for removal of PCR inhibitors. In our assay, The AMPure\u0026reg; beads purification was compared with the PowerClean Pro Cleanup Kit (Qiagen, Hilden, Germany) and displayed similar efficiency with respect to removal of PCR inhibitors (data not shown). The AMPure\u0026reg; clean-up was favoured as it is compatible with the prospect of automation of the whole analytical processing. One lingering question is whether positive results from qPCR systematically can be taken at value face as a proof of soil contamination, given that dead eggs of \u003cem\u003eToxocara\u003c/em\u003e can be associated with positive qPCR signals without also being able to cause toxocarosis when ingested by humans. Whereas no definitive answer for this question is available, the fact that \u003cem\u003eToxocara\u003c/em\u003e eggs can survive in the environment for several years [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and the probability that viable eggs can be mistaken for dead eggs upon light microscopic observation should prompt us to consider qPCR positive as a reliable marker of soil contamination with \u003cem\u003eToxocara\u003c/em\u003e eggs.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, the processing developed here and which combines a mechanical disruption of \u003cem\u003eToxocara\u003c/em\u003e eggs, DNA extraction using the DNeasy\u0026reg; PowerMax\u0026reg; Soil Kit and a subsequent DNA clean-up for removal of PCR inhibitors improves substantially detection of \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples by qPCR. Moreover, the potential for automation offered by this method can result in processing of hundreds of soil samples and allow to deliver timely results in a short period.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Wallonie-Bruxelles international agency (project 320167) with the contribution of the Dean of Faculty of Health Sciences, Poznań University of Physical Education.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJW, DJF, MWH and GJL conceived the study; JW, FWR and MWH provided eggs suspensions and environmental samples; JW carried out microscopic examination; DJF and WJ carried out molecular analyses; JW and DJF wrote the first draft of the paper, and JW, DJF, ILM, and JLG contributed to the final manuscript which they approve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge Tomasz Luty (DVM), PhD, for delivering \u003cem\u003eToxocara canis\u003c/em\u003e worms and J\u0026eacute;r\u0026ocirc;me Ambroise (CTMA), PhD, for his statistical analysis contributions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen J, Zhou DH, Nisbet AJ, Xu MJ, Huang SY, Li MW, et al. 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J Appl Microbiol 2012;113 5:1014\u0026ndash;26; doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2672.2012.05384.x\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pubmed/22747964\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Toxocara canis, Toxocara cati, helminth eggs; soil; sand; inhibitors; DNA extraction; qPCR; clean-up","lastPublishedDoi":"10.21203/rs.3.rs-264679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-264679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eToxocara canis\u003c/em\u003e (\u003cem\u003eT. canis\u003c/em\u003e)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eToxocara cati \u003c/em\u003e(\u003cem\u003eT. cati\u003c/em\u003e) are worldwide-distributed roundworms of canids and felids and causative agents of human toxocarosis, via ingestion of \u003cem\u003eToxocara\u003c/em\u003e eggs disseminated in the environment. Control of \u003cem\u003eToxocara \u003c/em\u003einfections is constrained by the lack of sensitive methods for screening of animal feces and environmental samples potentially contaminated by \u003cem\u003eToxocara\u003c/em\u003e eggs. We previously developed a quantitative duplex real-time PCR (qPCR) for sensitive and specific detection of \u003cem\u003eT. canis\u003c/em\u003e and \u003cem\u003eT. cati\u003c/em\u003e. In this work, a pre-analytical method for efficient extraction of DNA from \u003cem\u003eToxocara\u003c/em\u003e eggs present in environmental samples was set up. For this purpose, the influence of different methods for eggs lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eSoil and sand (10g) samples were spiked with egg suspensions of \u003cem\u003eT. canis\u003c/em\u003e. DNA was extracted from \u003cem\u003eToxocara\u003c/em\u003e eggs, using different DNA extraction kits (FastDNA™ SPIN Kit for Soil and DNeasy® PowerMax® Soil Kit), and an additional clean-up step (Agencourt® AMPure®). The efficiency of the above-developed process was compared with the conventional flotation-centrifugation and observation of \u003cem\u003eToxocara\u003c/em\u003e eggs under light microscopy.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe most effective DNA extraction method for \u003cem\u003eToxocara\u003c/em\u003e eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, DNA extraction with the DNeasy® PowerMax® Soil Kit and an additional DNA clean-up step with AMPure® beads. with a limit of detection of 6 eggs of \u003cem\u003eT. canis\u003c/em\u003e spiked in 10 g of soil with a probability of 97%.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe pre-analytical flow process developed here combined with qPCR represents an improved method for the surveillance of \u003cem\u003eToxocara \u003c/em\u003econtamination in the environment.\u003c/p\u003e","manuscriptTitle":"Optimized DNA-based Identification of Toxocara spp. Egg in Difficult Matrices: A Case for Specific and Sensitive Identification of Geohelminth Eggs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-25 21:57:39","doi":"10.21203/rs.3.rs-264679/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-04-20T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major Revision","date":"2021-04-20T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-04-02T00:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-17T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-02-25T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-02-25T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-23T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Parasites \u0026 Vectors","date":"2021-02-21T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-21T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-20T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2021-02-19T06:02:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6cc4579-91f7-427b-961a-43741ae6c91b","owner":[],"postedDate":"February 25th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2617692,"name":"Parasitology"}],"tags":[],"updatedAt":"2021-08-17T11:51:46+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-25 21:57:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-264679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-264679","identity":"rs-264679","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}