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. 2019:26:11.
doi: 10.1051/parasite/2019011. Epub 2019 Mar 6.

MALDI-TOF mass spectrometry: a new tool for rapid identification of cercariae (Trematoda, Digenea)

Antoine Huguenin  1 , Jérôme Depaquit  2 , Isabelle Villena  1 , Hubert Ferté  2
Affiliations

MALDI-TOF mass spectrometry: a new tool for rapid identification of cercariae (Trematoda, Digenea)

Antoine Huguenin et al. Parasite. 2019.

Abstract
in English, French

Identification of cercariae was long based on morphological and morphometric features, but these approaches remain difficult to implement and require skills that have now become rare. Molecular tools have become the reference even though they remain relatively time-consuming and expensive. We propose a new approach for the identification of cercariae using MALDI-TOF mass spectrometry. Snails of different genera (Radix, Lymnaea, Stagnicola, Planorbis, and Anisus) were collected in the field to perform emitting tests in the laboratory. The cercariae they emitted (Trichobilharzia anseri, Diplostomum pseudospathaceum, Alaria alata, Echinostoma revolutum, Petasiger phalacrocoracis, Tylodelphys sp., Australapatemon sp., Cotylurus sp., Posthodiplostomum sp., Parastrigea sp., Echinoparyphium sp. and Plagiorchis sp.) were characterized by sequencing the D2, ITS2 and ITS1 domains of rDNA, and by amplification using specific Alaria alata primers. A sample of each specimen, either fresh or stored in ethanol, was subjected to a simple preparation protocol for MALDI-TOF analysis. The main spectral profiles were analyzed by Hierarchical Clustering Analysis. Likewise, the haplotypes were analyzed using the maximum likelihood method. Analytical performance and the log-score value (LSV) cut-off for species identification were then assessed by blind testing. The clusters obtained by both techniques were congruent, allowing identification at a species level. MALDI-TOF enables identification at an LSV cut-off of 1.7 without false-positives; however, it requires more data on closely related species. The development of a "high throughput" identification system for all types of cercariae would be of considerable interest in epidemiological surveys of trematode infections.

L’identification des cercaires a longtemps été basée sur des caractéristiques morphologiques et morphométriques, mais ces approches restent difficiles à utiliser et nécessitent des compétences devenues rares de nos jours. Les outils moléculaires sont devenus la référence mais restent relativement coûteux en temps et en argent. Nous proposons une nouvelle approche pour l’identification des cercaires à l’aide de la spectrométrie de masse MALDI-TOF. Des mollusques de différents genres (Radix, Lymnaea, Stagnicola, Planorbis, Anisus) récoltés en milieu naturel ont été soumis à des tests d’émission au laboratoire. Les cercaires émises (Trichobilharzia anseri, Diplostomum pseudospathaceum, Alaria alata, Echinostoma revolutum, Petasiger phalacrocoracis, Tylodelphys sp., Australapatemon sp., Cotylurus sp., Posthodiplostomum sp., Parastrigea sp., Echinoparyphium sp. et Plagiorchis sp.) ont été caractérisées par séquençage des domaines D2, ITS2 et ITS1 de l’ADNr et par l’amplification d’amorces spécifiques d’Alaria alata. Un échantillon de chaque échantillon, frais ou stocké dans l’éthanol, a été soumis à un protocole de préparation simple pour l’analyse MALDI-TOF. Les spectres de référence obtenus (Main Spectra Profiles : MSP) ont été analysés par HCA (Hierarchical Clustering Analysis). Parallèlement, les haplotypes ont été analysés en Maximum de vraisemblance. La performance analytique et le seuil des valeurs de log-score (LSV) pour l’identification des espèces ont ensuite été évalués par des tests à l’aveugle. Les groupes obtenus par les deux techniques étaient congruents, permettant une identification à un niveau spécifique. Une valeur seuil de LSV à 1,7 a permis l’identification de cercaires en MALDI-TOF sans faux-positif, mais davantage de données sur les espèces étroitement apparentées sont nécessaires pour valider ce seuil. La mise au point d’un système d’identification “à haut débit” pour tous les types de cercaires présenterait un intérêt majeur pour les enquêtes épidémiologiques sur les trématodoses.

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Figures

Figure 1
Figure 1
Representative MALDI-TOF spectra of cercariae obtained using a simple direct-deposit protocol. Flex Analysis software, smoothed spectra with baseline substracted A: Trichobilharzia anseri (FODR1) B: Diplostomum pseudospathaceum (LFDR1) C: Tylodelphys sp. (LFZE1) D: Australapatemon sp. (LFDC42) E: Cotylurus sp. (LFDC89) F: Posthodiplostomum sp. (LFDC83) G: Alaria alata (LFDC 41) H: Echinoparyphium sp. (ECDC26) I: Echinostoma revolutum (ECJO3) J: Petasiger phalacrocoracis (ECDR1) K: Plagiorchis sp. (XIDR3)
Figure 2
Figure 2
Panel A: Hierarchical clustering dendrogram of MALDI-TOF MSP, using the correlation distance measure and Ward algorithm. All the MSPs were included in the final database. Panel B: Maximum likelihood tree of the D2 domain of the cercariae strains present in the MALDI-TOF spectral database.
Figure 3
Figure 3
ROC curve for determination of the optimal LSV cut-off. Logistic regression model based on identification prediction using the final version of the MALDI-TOF cercariae database compared to molecular identification.
Figure 4
Figure 4
Panel A: Representative MALDI-TOF spectra of the same specimen (LFDC53) under the following conditions: fresh cercariae, ethanol fixation, and ethanol conservation (12 months). Panel B: Superposition of panel A spectra.
See this image and copyright information in PMC

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