Loop-mediated isothermal amplification as a reliable assay for Toxocara canis infection in pet dogs

Abstract

Keeping of infected dogs as pet results in the potential transmission risk factors for shedding helminthic infections such as toxocariasis. Lack of accurate identification of Toxocara canis eggs in non-dewormed infected pet dogs remains a diagnostic concern among researchers. In this study, dog owners were asked to fill up a questionnaire regarding their pets and their attitude towards the deworming regimen. One hundred faecal samples were collected from pet dogs (Northwest Iran) and were subsequently identified by the ZnSo4 flotation technique, PCR and loop-mediated isothermal amplification (LAMP) assays. The DNA of the recovered T. canis eggs was then extracted and amplified by LAMP and PCR. Furthermore, ITS2 amplicons were sequenced for appraisal of the phylogenetic analysis. Nine, 5 and 11% of T. canis infections were identified by microscopy, PCR and LAMP, respectively. It was detected that LAMP was 10 times (10to 10 g/μl) more sensitive than PCR (10to 10 g/μl). The kappa value between LAMP and PCR indicated a faint concurrence (0.463). The kappa coefficient between LAMP and flotation technique indicated a strong agreement (0.667). The highest infection rate (n = 11) was detected in non-dewormed pet dogs, particularly those less than 3 months old (P < 0.05). None of the infected dogs had a history of walking and kennelled behaviours in public places. The LAMP assay can address as a simple, rapid and highly sensitive technique for detecting low burden of T. canis eggs in infected pet dogs. It was proposed that the dog holder's awareness is insufficient to implement regular deworming schedules. Additionally, regional policymakers should broadly revise anthelmintic treatment guidelines.

Detection of helminths by loop-mediated isothermal amplification assay: a review of updated technology and future outlook

“…The impressive progress currently made in the LAMP assay for helminths includes trematodes of Clonorchis sinensis [12, 26, 60], Opisthorchis viverrini [14, 61, 62], Amphimerus spp. [63, 64], Paragonimus westermani [15], Fasciola hepatica [65–67], F. gigantica [65], Schistosoma japonicum [16, 27, 68–70], S. mansoni [13, 71–77], S. haematobium [51, 71, 72, 76]; nematodes of Necator americanus [78, 79], Ascaris lumbricoides [17, 79] , Trichuris trichiura [79], Toxocara canis [80] and T. cati [81], Strongyloides stercoralis [52, 82], Onchocerca volvulus [83–86], Wuchereria bancrofti [86, 87], Brugia malayi [86, 88], B. tomori [88], Loa loa [89–91], Dirofilaria repens [92], Angiostrongylus cantonensis [93, 94], Trichinella spiralis [95, 96], Bursaphelenchus xylophilus [97], and Haemonchus contortus [98, 99]; cestodes of T. solium [44, 100–103] , T. saginata [44, 100–103], T. asiatica [44, 100–103], T. hydatigena [104], T. multiceps [104], T. pisiformis [104] and T. crassiceps [104], Echinococcus granulosus [104–106], E. multilocularis [104, 107], E. equinus [108], E. canadensis [108], E. felidi [108], E. ortleppi [108, 109] and E. shiquicus [104] , have been covered in this review for further insight into its adoption for clinical diagnosis, field surveys and surveillance of helminths. The sensitivity and specificity of...…”

“…pseudospiralis and Tri. nelsoni , no cross reaction with heterologous species, no template-free amplification[95]0.002 larvae/rxn0.01 larvae/g of muscle tissuemt-lsrDNAGU339148.10.1 pg/rxn100%[96] T. canis Egg––ITS-2AJ002440,0.1 pg of DNA/rxn100%[81]EggSand–3 eggs/10 g of sandAdultStoolDog0.1 pg of DNA/rxn100%[80]3 eggs/30 g of stools T. catti EggSand–ITS-2AJ0024410.1 pg/rxn100%[81] B. xylophilus –––ITSAB500146- AB500156(accessed in DDBJ)10 copies of DNA/rxn100%[97]–Wood–2.5 × 10^(−5) of a nematode/rxn– H. contortus EggStoolSheepITS-1–5 pg of DNA/rxn100%[98]Adult–GoatITS-2X78803.11 pg of DNA/rxn100%[99]Cestodes T. solium Proglottid and cysticercusCyst fluidMouseCox1AB086256–100%[100]Proglottid and cysticercusCyst fluidMouseClpAB4418151 copy of DNA/rxn100% T. saginata Proglottid and cysticercusCyst fluidMouseCox1AY684274–100%[100]EggStoolHuman5 EPG100%Proglottid and cysticercusCyst fluidMouse…”

“…The prevention of its transmission depends on the condition of the environmental contamination levels and the accurate determination of its sources [81]. However, Toxocara identification by traditional microscopy of stools from pets or environmental samples remains a methodological concern due to its insensitivity in low-burden cases and its difficulty in distinguishing T. canis from T. cati eggs [80, 81]. PCR assays have been designed to discern Toxocara spp.…”

“…The species-specific LAMP assay targeting ITS-2 was validated by two groups and found to be ten-fold more sensitive than PCR without cross reactivity in the laboratory between Toxocara spp. and is applied in domesticated dogs and sand samples [80, 81]. In the context of environmental specimens, LAMP manifests a detection limit of 3 eggs/10 g of sand and less than 3 eggs/30 g of stools, compared with the 6 eggs/10 g of sand and more than 2 eggs/30 g of stools detection limit of PCR [80, 81].…”

“…and is applied in domesticated dogs and sand samples [80, 81]. In the context of environmental specimens, LAMP manifests a detection limit of 3 eggs/10 g of sand and less than 3 eggs/30 g of stools, compared with the 6 eggs/10 g of sand and more than 2 eggs/30 g of stools detection limit of PCR [80, 81]. In a field survey of soil contamination, LAMP yield a positive rate of 42.7% versus 7.7% of PCR [157].…”