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

Durant, Jean-François; Irenge, Léonid M.; Fogt-Wyrwas, Renata; Dumont, Catherine; Doucet, Jean‐Pierre; Mignon, Bernard; Losson, Bertrand; Gala, Jean‐Luc

doi:10.1186/1756-3305-5-288

Cited by 62 publications

(61 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…PCR has served to enable molecular characterization of faecal samples of dogs and cats as well as soil containing eggs of Toxocara spp. (FOGT-WYRWAS et al, 2007;FAHRION et al, 2011;DURANT et al, 2012;KHADEMVATAN et al, 2013KHADEMVATAN et al, , 2014. PCR technique was applied in an experimental study to evaluate bronchoalveolar lavage (PINELLI et al, 2013) and also to confirm the diagnosis of human toxocariasis from CSF samples to isolate the cause of neurological syndrome (CALDERA et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Limit of detection of Toxocara canis larvae in experimentally contaminated bovine milk

et al. 2016

View full text Add to dashboard Cite

This study aimed to evaluate the limit of detection of Toxocara canis larvae in experimentally contaminated commercial bovine milk samples, based on a centrifuge-sedimentation technique. Firstly, bovine milk (whole and skim) samples were contaminated with 50 T. canis larvae in order to evaluate the interference of milk fat with the recovery of the larvae. Next, the effects of 10% formalin (100 µL), ether (100 µL), and a combination of both solutions on the recovery of the larvae was examined. Thereafter, the limit of detection of the larvae was determined using the solution (from step 2) considered optimal for degreasing the milk sample. Samples were contaminated with aliquots of 1, 5, 10, 25, and 50 larvae. For each milk sample (1.0 mL), 15 repetitions were analysed. The recovery of the larvae from the skim milk samples was higher (p = 0.0031) than that from the whole milk samples. No significant difference (p = 0.5681) was observed with regard to the percentage of recovered larvae when comparing the degreasing solutions. Nevertheless, the formalin-ether combination was more efficient for recovering the larvae (73.1%) than ether (71.9%), formalin (67.6%), and pure whole milk (70.0%). Concerning the limit of detection (using formalin-ether), all the samples contaminated with 5, 10, 25, and 50 larvae tested positive (minimum: 62.7%). Of the samples contaminated with a single larva, 66.7% tested positive. These results suggest that the centrifugation-sedimentation technique may be useful for recovering larvae of Toxocara spp. in naturally or experimentally contaminated milk samples obtained from a wide range of animal species. Key words: Toxocariasis, recovery of larvae, diagnosis ResumoO objetivo do estudo foi avaliar o limite de detecção de larvas de Toxocara canis em leite bovino comercial contaminado experimentalmente, a partir de uma técnica de centrifugo-sedimentação. Primeiramente, amostras de leite bovino comercial (integral e desnatado) foram contaminadas com 50 larvas de T. canis, para avaliação da interferência da gordura sobre a recuperação das larvas. Em uma segunda etapa, foi avaliada a ação de formalina 10% (100 µL), éter (100 µL) e combinação das soluções. A terceira etapa consistiu da verificação do limite de detecção de larvas, com uso da melhor solução desengordurante (etapa 2), em amostras de leite (1,0 mL) contendo 1; 5; 10; 25 e 50 larvas. Para cada análise de detecção do leite (1,0 mL), foram realizadas 15 repetições. Foi observado que o percentual de recuperação de larvas no leite desnatado foi significativamente maior (p= 0,0031) que o observado no leite integral. Na comparação das soluções, não houve diferença significativa (p= 0,5681) no percentual de larvas recuperadas. Entretanto, houve uma maior recuperação quando do emprego da combinação de formol-éter (73,1%) em relação ao éter (71,9%), ao formol (67,6%) e ao leite integral puro (70,0%). Em relação ao limite de detecção, com uso de formalina-éter, todas as alíquotas apresentaram resultado positivo (mínimo de 62,7% de larvas ...

show abstract

Section: Discussionmentioning

confidence: 99%

Limit of detection of Toxocara canis larvae in experimentally contaminated bovine milk

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Thanks to these advanced techniques, zoonotic parasites such as Toxocara spp. (21) and Echinococcus multilocularis (22,23) can be detected and quantified in stools. Real-time nested PCR using fluorescence resonance energy transfer (FRET) probes was designed to detect and discriminate between different host species by melting curve analysis for field studies focusing on Echinococcus multilocularis investigations and the carnivore hosts Vulpes vulpes (red fox), Vulpes ferrilata (Tibetan fox), and Canis lupus familiaris/Canis lupus (domestic dog/wolf) (22).…”

mentioning

confidence: 99%

“…However, to date, very few sensitive tools have been developed to describe environmental contamination by Toxocara spp. (21) and to assess the respective roles of their definitive hosts in spreading these organisms.…”

mentioning

confidence: 99%

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

Knapp

Umhang

Poulle

et al. 2016

Appl Environ Microbiol

View full text Add to dashboard Cite

dStudying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes. With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. For detection of infectious agents in the environment, which involves both wild and domestic animals, coprosamples are noninvasive and valuable isolates, as opposed to necropsy, which is time-consuming, logistically onerous, and often considered unethical by the public. In this field of work, Echinococcus multilocularis is the causative agent of alveolar echinococcosis, one of the most worrying zoonoses in the Northern Hemisphere (1), and is in constant progression (2-4). Toxocara spp. are the causative agents of toxocariasis, a widespread disease that is still described in known areas of endemicity despite relevant anthelminthic programs (5). Both E. multilocularis and Toxocara spp. are zoonotic agents involved in a fecal-oral transmission cycle from carnivores to humans, with no vectors or intermediate actors (5). These helminth parasites are both found in at least one wild carnivore (red fox) and two domestic carnivore (dog and cat) host species. While E. multilocularis is a common tapeworm of the red fox, low parasite prevalences have often been described for European domestic host species (cat and dog) (6-8). E. multilocularis eggs are highly resistant to environments with low temperatures and high percentages of relative humidity. Under controlled laboratory conditions, the eggs were found to survive in water for 478 days at 4°C. Under these experimental conditions, eggs remained infectious for rodents (9). Carnivores become infected after predation of contaminated rodents and release eggs into the environment via their feces. Alveolar echinococcosis in humans remains a let...

show abstract

“…canis and T . cati eggs in fecal and soil samples [ 27 ]. Human stool microscopy is of no benefi t because Toxocara species do not complete their life cycle in humans.…”

Section: Diagnosismentioning

confidence: 99%

The Diagnosis and Classification of Parasitic Diseases of the Liver

Mandal

2013

Liver Immunology

View full text Add to dashboard Cite

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

Cited by 62 publications

References 24 publications

Limit of detection of Toxocara canis larvae in experimentally contaminated bovine milk

Limit of detection of Toxocara canis larvae in experimentally contaminated bovine milk

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

The Diagnosis and Classification of Parasitic Diseases of the Liver

Contact Info

Product

Resources

About