PCR-Based Detection ofAngiostrongylus cantonensisin Tissue and Mucus Secretions from Molluscan Hosts

Abstract

Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.

Spontaneous Shedding of Metastrongyloid Third-Stage Larvae by Experimentally Infected Limax maximus

“…Release of L3 via gastropod faeces has not been reported previously. Detection of metastrongyloid L3 in mucus trails or exiting directly through the body surface of slugs has been reported (Ash, 1976, Bonetti et al 1998, Heyneman and Lim 1967, Jenkins et al 2006, Kralka and Samuel 1984, Kutz et al 2000, Qvarnstrom et al 2007. No L3 were detected in the mucus of A. cantonensis infected Limax flavus (Campbell and Little, 1988).…”

“…Recently gastropod L3 shedding has also been reported for a crenosomatid (Troglostrongylus brevior) (Giannelli et al 2015). Detection of L3 in mucus of slugs and semi-slugs has been reported for A. cantonensis and A. costaricensis, although in small numbers and able to survive for only short periods of time outside the gastropod host, bringing into question what role it may play as a source of exposure in natural infections (Ash, 1976, Bonetti et al 1998, Cowie, 2013, Prociv et al 2000, Qvarnstrom et al 2007). L3 of A. cantonensis may also be released by land snails and slugs immersed in water suggesting that contaminated drinking water could be a potential route of exposure for human infection (Cheng and Alicata, 1964).…”

Hurdles in the evolutionary epidemiology of Angiostrongylus cantonensis: Pseudogenes, incongruence between taxonomy and DNA sequence variants, and cryptic lineages

“…Indeed, the use of DNA data only came about in the 2000s. For example, ribosomal DNA sequences were used to assess metastrongylid nematode relationships (Carreno & Nadler, 2003) or to help survey larvae from mollusk intermediate hosts (Fontanilla & Wade, 2008; Qvarnstrom, Sullivan, Bishop, Hollingsworth, & da Silva, 2007; Qvarnstrom et al., 2010), and ~360‐bp region of the cytochrome c oxidase 1 ( CO 1) gene was used to assess relationships to other species of Angiostrongylus (Eamsobhana et al., 2010). To date, molecular systematic/phylogeographic studies on A. cantonensis have mainly used two mitochondrial (mtDNA) markers, CO 1 and cytochrome b ( CYTB ), where the focus has largely been descriptive in terms of reporting local patterns of haplotype variants (Aghazadeh et al., 2015; Dalton, Fenton, Cleveland, Elsmo, & Yabsley, 2017; Dusitsittipon, Criscione, Morand, Komalamisra, & Thaenkham, 2017; Dusitsittipon, Thaenkham, Watthanakulpanich, Adisakwattana, & Komalamisra, 2015; Eamsobhana, Song, et al., 2017; Eamsobhana, Yong, et al., 2017; Lv et al., 2012; Monte et al., 2012; Moreira et al., 2013; Nakaya et al., 2013; Okano et al., 2014; Rodpai et al., 2016; Simoes et al., 2011; Tokiwa et al., 2012, 2013; Vitta et al., 2016; Yong, Eamsobhana, Song, Prasartvit, & Lim, 2015; Yong, Song, Eamsobhana, Goh, & Lim, 2015; Yong, Song, Eamsobhana, & Lim, 2016).…”

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

“…Dentro del molusco, la larva se desarrolla en las etapas L 2 y L 3 , para luego abandonar el huésped invertebrado (10). Además, el nematodo tiene huéspedes paraténicos, como crustáceos, sapos y planarias, que facilitan su dispersión a diversos ecosistemas donde habitan mamíferos, aves y personas, los cuales son huéspedes accidentales en riesgo de contraer la enfermedad (9,11).…”

“…En las personas ocasiona meningitis eosinofílica, generada por las larvas L 3 procedentes de los caracoles, las cuales ingresan en los pacientes por la vía oral, penetran la pared intestinal y viajan por el torrente sanguíneo hasta el cerebro, donde causan daño tisular y reacción inflamatoria (9,12). La sintomatología es inespecífica, y depende de la ubicación y el número de larvas que ingresan al cerebro, donde puede generar dolor de cabeza, alteraciones neurológicas, coma y la muerte (7).…”