Detection of Spiked Fasciola hepatica Eggs in Stool Specimens Using LAMP Technique
Background: Fascioliasis is one of the most important food-borne worm disease caused by Fasciola sp. Parasitological diagnosis is more difficult due to the low parasite burden and a few eggs shedding of helminths. Therefore, it will be valuable to development of simple, fast and reliable diagnostic tests for detection of human and animal fascioliasis.
Methods: Infected liver collected from abattoir in Tehran, Iran in 2017. F. hepatica eggs were detached from the uterus of worms under a stereo microscope. Various numbers of eggs were spiked to 200 mgr. of negative feces samples. DNA was extracted and then target regions (nuclear IGS) were amplified by LAMP assay using six primers. Fecal specimens without egg and DNA of other helminths were used as negative controls. F. hepatica sample which confirmed by morphologic criteria and PCR- RFLP was used as positive control.
Results: LAMP products by using SYBR Green I could detect even a single egg in fecal samples which was visible by change of color from orange to green. There was no cross amplification by other helminths including; Taenia saginata, Dicrosolium dendriticum and F. gigantica.
Conclusion: LAMP seems a rapid, sensitive, cost-effective technique for detection of human fascioliasis.
Mas-Coma S. Epidemiology of fascioliasis in human endemic areas. Journal of helminthology. 2005;79(3):207-16.
Reaghi S, Haghighi A, Harandi MF, Spotin A, Arzamani K, Rouhani S. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran. Veterinary world. 2016;9(9):1034-8.
Rouhani S, Raeghi S, Spotin A. Spermatogenic and Phylo-molecular Characterizations of Isolated Fasciola Spp. From Cattle, North West Iran. Pakistan journal of biological sciences : PJBS. 2017;20(4):204-9.
Mas-Coma S, Bargues MD, Valero MA. Fascioliasis and other plant-borne trematode zoonoses. International journal for parasitology. 2005;35(11-12):1255-78.
Nguyen TG, Van De N, Vercruysse J, Dorny P, Le TH. Genotypic characterization and species identification of Fasciola spp. with implications regarding the isolates infecting goats in Vietnam. Experimental parasitology. 2009;123(4):354-61.
Le TH, De NV, Agatsuma T, Thi Nguyen TG, Nguyen QD, McManus DP, et al. Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam. International journal for parasitology. 2008;38(6):725-30.
Itagaki T, Kikawa M, Terasaki K, Shibahara T, Fukuda K. Molecular characterization of parthenogenic Fasciola sp. in Korea on the basis of DNA sequences of ribosomal ITS1 and mitochondrial NDI gene. The Journal of veterinary medical science / the Japanese Society of Veterinary Science. 2005;67(11):1115-8.
Itagaki T, Sakaguchi K, Terasaki K, Sasaki O, Yoshihara S, Van Dung T. Occurrence of spermic diploid and aspermic triploid forms of Fasciola in Vietnam and their molecular characterization based on nuclear and mitochondrial DNA. Parasitology international. 2009;58(1):81-5.
Rouhani S, Raeghi S, Mirahmadi H, Harandi MF, Haghighi A, Spotin A. Identification of Fasciola spp. in the East of Iran, Based on the Spermatogenesis and Nuclear Ribosomal DNA (ITS1) and Mitochondrial (ND1) Genes. Archives of Clinical Infectious Diseases. 2017;12(2):e57283.
Arifin MI, Hoglund J, Novobilsky A. Comparison of molecular and conventional methods for the diagnosis of Fasciola hepatica infection in the field. Veterinary parasitology. 2016;232:8-11.
Mezo M, Gonzalez-Warleta M, Carro C, Ubeira FM. An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). The Journal of parasitology. 2004;90(4):845-52.
Brockwell YM, Spithill TW, Anderson GR, Grillo V, Sangster NC. Comparative kinetics of serological and coproantigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole. Veterinary parasitology. 2013;196(3-4):417-26.
Fairweather I. Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy? Veterinary parasitology. 2011;180(1-2):133-43.
Fohse L, Suffner J, Suhre K, Wahl B, Lindner C, Lee CW, et al. High TCR diversity ensures optimal function and homeostasis of Foxp3+ regulatory T cells. European journal of immunology. 2011;41(11):3101-13.
Martinez-Valladares M, Rojo-Vazquez FA. Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions. Parasites & vectors. 2016;9:73.
Ai L, Li C, Elsheikha HM, Hong SJ, Chen JX, Chen SH, et al. Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay. Veterinary parasitology. 2010;174(3-4):228-33.
Salant H, Abbasi I, Hamburger J. The development of a loop-mediated isothermal amplification method (LAMP) for Echinococcus granulosus [corrected] coprodetection. The American journal of tropical medicine and hygiene. 2012;87(5):883-7.
Fernandez-Soto P, Gandasegui Arahuetes J, Sanchez Hernandez A, Lopez Aban J, Vicente Santiago B, Muro A. A loop-mediated isothermal amplification (LAMP) assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model. PLoS neglected tropical diseases. 2014;8(9):e3126.
Spithill TW, Dalton JP. Progress in development of liver fluke vaccines. Parasitol Today. 1998;14(6):224-8.
Bendezu P, Frame A, Hillyer GV. Human fascioliasis in Corozal, Puerto Rico. The Journal of parasitology. 1982;68(2):297-9.
Knobloch J, Delgado E, Alvarez A, Reymann U, Bialek R. Human fascioliasis in Cajamarca/Peru. I. Diagnostic methods and treatment with praziquantel. Trop Med Parasitol. 1985;36(2):88-90.
Mas-Coma MS, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bulletin of the World Health Organization. 1999;77(4):340-6.
Magalhaes KG, Jannotti-Passos LK, Caldeira RL, Berne ME, Muller G, Carvalho OS, et al. Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR. Veterinary parasitology. 2008;152(3-4):333-8.
Cucher MA, Carnevale S, Prepelitchi L, Labbe JH, Wisnivesky-Colli C. PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails. Veterinary parasitology. 2006;137(1-2):74-82.
Arimatsu Y, Kaewkes S, Laha T, Sripa B. Specific diagnosis of Opisthorchis viverrini using loop-mediated isothermal amplification (LAMP) targeting parasite microsatellites. Acta tropica. 2015;141(Pt B):368-71.
Ni XW, McManus DP, Lou ZZ, Yang JF, Yan HB, Li L, et al. A comparison of loop-mediated isothermal amplification (LAMP) with other surveillance tools for Echinococcus granulosus diagnosis in canine definitive hosts. PloS one. 2014;9(7):e100877.
Karthik K, Rathore R, Thomas P, Arun TR, Viswas KN, Dhama K, et al. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination. MethodsX. 2014;1:137-43.
Zhou D, Guo J, Xu L, Gao S, Lin Q, Wu Q, et al. Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane. Scientific reports. 2014;4:4912.
Zhang X, Liao M, Jiao P, Luo K, Zhang H, Ren T, et al. Development of a loop-mediated isothermal amplification assay for rapid detection of subgroup J avian leukosis virus. Journal of clinical microbiology. 2010;48(6):2116-21.
Thekisoe OM, Bazie RS, Coronel-Servian AM, Sugimoto C, Kawazu S, Inoue N. Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates. The Journal of veterinary medical science. 2009;71(4):471-5.
Ni X, McManus DP, Yan H, Yang J, Lou Z, Li H, et al. Loop-mediated isothermal amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts. Parasites & vectors. 2014;7:254.
|Issue||Vol 14 No 3 (2019)|
|LAMP Fasciola hepatica Spiked egg|
|Rights and permissions|
|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.|