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

Parasites and Vectors - 2021
Wojciech Jarosz1, Jean-François Durant2, Léonid M. Irenge2, Renata Fogt-Wyrwas1, Hanna Mizgajska-Wiktor1, Jean‐Luc Gala2
1Department of Biology and Anatomy, Faculty of Health Sciences, Poznań University of Physical Education, Królowej Jadwigi 27/39, 61-871, Poznan, Poland
2Center for Applied Molecular Technologies, Institute of Clinical and Experimental Research, Université Catholique de Louvain, Tour Claude Bernard, Avenue Hippocrate 54-55, 1st Floor, B1.54.01, 1200, Brussels, Belgium

Tóm tắt

Abstract 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. Graphical Abstract

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Tài liệu tham khảo

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;12:1344–8.

Holland CV. Knowledge gaps in the epidemiology of Toxocara: the enigma remains. Parasitology. 2017;144:81–94.

Chen J, Liu Q, Liu GH, Zheng WB, Hong SJ, Sugiyama H, et al. Toxocariasis: a silent threat with a progressive public health impact. Infect Dis Poverty. 2018;7:59.

Macpherson CN. The epidemiology and public health importance of toxocariasis: a zoonosis of global importance. Int J Parasitol. 2013;43:999–1008.

Overgaauw PA. Aspects of Toxocara epidemiology: human toxocarosis. Crit Rev Microbiol. 1997;23:215–31.

Durant JF, Irenge LM, Fogt-Wyrwas R, Dumont C, Doucet JP, Mignon B, et al. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples. Parasit Vectors. 2012;5:288.

Tyungu DL, McCormick D, Lau CL, Chang M, Murphy JR, Hotez PJ, et al. Toxocara species environmental contamination of public spaces in New York City. PLoS Negl Trop Dis. 2020;14:e0008249.

Amoah ID, Singh G, Stenstrom TA, Reddy P. Detection and quantification of soil-transmitted helminths in environmental samples: a review of current state-of-the-art and future perspectives. Acta Trop. 2017;169:187–201.

Umhang G, Bastien M, Renault C, Faisse M, Caillot C, Boucher JM, et al. A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR. Parasite. 2017;24:28.

Mizgajska H. Eggs of Toxocara spp. in the environment and their public health implications. J Helminthol. 2001;75:147–51.

Mizgajska-Wiktor H. Recommended method for recovery of Toxocara and other geohelminth eggs from soil. Wiad Parazytol. 2005;51:21–2.

Pabinger S, Rodiger S, Kriegner A, Vierlinger K, Weinhausel A. A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomol Detect Quantif. 2014;1:23–33.

Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29(Suppl 1):S49-52.

Phasuk N, Kache R, Thongtup K, Boonmuang S, Punsawad C. Soil contamination with Toxocara eggs in public schools in rural areas of southern Thailand. J Trop Med. 2020;2020:9659640.

Azam D, Ukpai OM, Said A, Abd-Allah GA, Morgan ER. Temperature and the development and survival of infective Toxocara canis larvae. Parasitol Res. 2012;110:649–56.

Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 2012;113:1014–26.

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