[Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum].

INTRODUCTION: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. OBJECTIVE: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. MATERIALS AND METHODS: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. RESULTS: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). CONCLUSIONS: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Estandarización de una prueba múltiple de reacción en cadena de la polimerasa en tiempo real para la identificación de Angiostrongylus cantonensis, A. costaricensis y A. vasorum / Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Resumen Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específica para el manejo de las angiostrongilosis humana y la animal. Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus. Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®. Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano. Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %). Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

Abstract Introduction: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. Objective: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. Materials and methods: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. Results: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). Conclusions: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Diagnosing and Understanding Angiostrongyliasis, A Zoonotic Cause of Meningitis.

Eosinophilic meningitis caused by Angiostrongylus cantonensis is spreading worldwide, and it can manifest as a severe neurological disease. Angiostrongyliasis is a food- and water-borne parasitosis that usually exhibits a seasonal and circumscribed geographical distribution. To improve control and treatment of these infections, further studies of transmission dynamics under natural conditions and the development of better diagnostic tools and treatment options are needed.

First report of the activity of predatory fungi on Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) first-stage larvae.

The nematode Angiostrongylus cantonensis causes eosinophilic meningoencephalitis in humans and thus alternative methods of control should be studied. The objective of this work was to evaluate the predatory capacity of eight fungal isolates of the species Duddingtonia flagrans (AC001, CG768 and CG722), Monacrosporium thaumasium (NF34), M. sinense (SF53) and Arthrobotrys robusta (I31), A. cladodes (CG719) and A. conoides (I40) on first-stage larvae (L1) of A. cantonensis under laboratory conditions. The treated groups contained 1000 conidia of the fungal isolates and 1000 A. cantonensis L1 in Petri dishes containing 2% water-agar medium (2% WA). The control group (without fungi) contained only 1000 A. cantonensis L1 in 2% WA. Evidence of predation was observed at the end of 7 days. Percentage reductions in L1 were: AC001, 82.8%; CG768, 71.0%; CG722, 72.8%; NF34, 86.7%; SF53, 89.7%; I40, 48.3%; CG719, 84.7%; and I31, 80.4%. No significant difference was observed (p>0.01) between the actions of the isolates used; however, a difference was noted (p<0.01) in relation to the control group. The results of the present work, confirm previous reports of the effectiveness of the fungi D. flagrans, M. thaumasium, M. sinense and A. robusta in controlling larvae of potentially zoonotic nematodes, this being the first report on A. cantonensis L1.

Molecular evidence for the endosymbiont Wolbachia in a non-filaroid nematode, Angiostrongylus cantonensis.

Wolbachia harbored by most filarial parasites, is critical to both embryogenesis and microfilarial development, and may lead to inflammation and pathogenesis in infected hosts. Based on alignment of the sequences from the wsp, ftsZ, and 16S rRNA genes, Wolbachia was demonstrated to exist in Angiostrongylus cantonensis, a non-filaroid nematode. Although the wsp gene may not be the best candidate for evolutionary analysis of Wolbachia, this gene has been sequenced from a broader coverage of the host species, making it feasible to be used for phylogenetic analysis in this study. The results from both Neighbor-joining and Maximum parsimony methods showed that this novel Wolbachia does not belong to any of the known groups (C or D) of nematode-derived Wolbachia. In addition, the wsp gene sequence of this newly identified endosymbiont revealed a high degree of identity (98%) with that from Diaea circumlita c2, tentatively classified into the putative group G. This suggests that Wolbachia from A. cantonensis could represent a deeply branched lineage in Wolbachia evolution or the occurrence of horizontal transfer between infected hosts. In conclusion, the findings provide some insights into our understanding of the evolution of Wolbachia, particularly the isolate from A. cantonensis.