A multiplex PCR protocol for rapid differential identification of four families of trematodes with medical and veterinary importance transmitted by Biomphalaria Preston, 1910 snails

Mesquita, Silvia Gonçalves; Rodrigues-Luiz, Gabriela F.; Reis-Cunha, João Luís; Cardoso, Mariana Santos; Mendonça, Cristiane Lafetá Furtado; Bueno, Lilian Lacerda; Fujiwara, Ricardo Toshio; Pinto, Hudson Alves; Caldeira, Roberta Lima; Bartholomeu, Daniella Castanheira

doi:10.1016/j.actatropica.2020.105655

Cited by 7 publications

(9 citation statements)

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“…Trematode infection in snails was investigated using a multiplex PCR protocol that enables the differentiation of four important families commonly found parasitizing Biomphalaria snails [ 38 , 54 – 56 ]. Schistosomatidae species were detected in 44.4% (8/18) of the study sites, while Echinostomatidae and Strigeidae were each found in 5.5% (1/18) of the sites.…”

Section: Discussionmentioning

confidence: 99%

“…In order to investigate the presence of trematode infection, the gDNA obtained from snail samples from all collection sites was used as the template for a trematode family-specific multiplex PCR in accordance with Mesquita et al [ 38 ]. To compare the size of the amplified DNA fragments obtained from the field material, various positive controls were included using gDNA from cercariae belonging to the following trematode families: Clinostomidae, Echinostomatidae, Schistosomatidae, and Strigeidae.…”

Section: Methodsmentioning

confidence: 99%

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A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

et al. 2021

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Background Schistosomiasis a neglected tropical disease endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. Methods Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. Results Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. Conclusions Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques. Graphical abstract

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

et al. 2021

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“…The presence of trematode infection in snails was investigated using a multiplex PCR protocol that enables the differentiation of four important families commonly found parasitizing Biomphalaria snails (37,(60)(61)(62). Schistosomatidae species were detected in 44.4% (8/18) of the study sites, while Echinostomatidae and Strigeidae were each found in 5.5% (1/18).…”

Section: Discussionmentioning

confidence: 99%

“…In order to investigate the presence of trematode infection, the gDNA obtained from snail samples from all collection sites were used as template for a trematode-family-speci c multiplex PCR according to Mesquita et al (37). The gDNA from isolated cercariae were also used to con rm morphological identi cation.…”

Section: Multiplex Pcr For Family-speci C Molecular Identi Cation Of Trematodesmentioning

confidence: 99%

Optimization of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Schistosoma Mansoni (Trematoda: Digenea) Detection in Biomphalaria spp. from Endemic Areas for Schistosomiasis in Brazil

Mesquita

Neves

Scholte

et al. 2021

Preprint

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Background: Schistosomiasis mansoni is a neglected tropical disease endemic in Brazil caused by Schistosoma mansoni, which is transmitted by Biomphalaria snails. Among all measures to control and eliminate the disease, accurate mapping and monitoring of snail breeding sites for susceptible and/or infected hosts in endemic areas are recommended. Parasitological methods are frequently used to identify infected snails, although they have many limitations, often providing false-negative results. Loop-mediated isothermal amplification (LAMP) is a promising alternative method for a more sensitive, rapid, and cost-effective diagnosis. However, standardization of LAMP assays is challenging due to the variety of parasites that are co-endemic with S. mansoni, and their varying prevalence rates in different areas. In this work, we aimed to optimize a LAMP assay for the detection of S. mansoni in Biomphalaria snails from endemic areas in the state of Minas Gerais, Brazil. Methods: A total of 1,001 snails were collected in five municipalities of the Mucuri and Jequitinhonha Valleys. Snails were pooled and squeezed according to the collection site to detect the presence of the larval forms of S. mansoni and other trematodes. Pooled snails were submitted to pepsin digestion and DNA extraction. Then molecular assays were performed for the species-specific identification and characterization of the samples. A LAMP assay was optimized and validated using laboratory and field samples. Results: Using the parasitological method, S. mansoni cercariae were detected in snails from two collection sites. Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites by PCR-RFLP. Multiplex PCR, LS-PCR, and conventional PCR allowed the detection of positive snails in four additional sites. The optimized LAMP assay was effective in detecting the presence of S. mansoni infection with 100% sensitivity, 91.66% specificity, and a Kappa index of 0.88, when compared to LS-PCR and conventional PCR. Conclusions: Our findings suggest that LAMP is a good alternative for the detection and monitoring of transmission foci of S. mansoni, as it enabled the detection of infection three times more than the parasitological examination and is more applicable directly in the field when compared to other molecular approaches.

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“…Compared to shedding, these tend to be more sensitive for detecting infections (an additional 10%–60%; Born‐Torrijos et al, 2014) and they allow cost‐efficient screening of large snail populations when combined with a pooling design (Carolus et al, 2019). However, they typically target a single species or limited set of species, usually those of medical or veterinary importance (e.g., Caron et al, 2011; Hamburger et al, 2004; Jannotti‐Passos et al, 2006; Mesquita et al, 2020; Pennance et al, 2020; Schols et al, 2019). The ability of xenomonitoring PCRs to detect co‐infections is therefore limited to a very restricted set of target species (e.g., Mesquita et al, 2020), making these methods inappropriate for trematode community studies.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous genotyping of snails and infecting trematode parasites using high‐throughput amplicon sequencing

Hammoud

Mulero

Bocxlaer

et al. 2021

Molecular Ecology Resources

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A multiplex PCR protocol for rapid differential identification of four families of trematodes with medical and veterinary importance transmitted by Biomphalaria Preston, 1910 snails

Cited by 7 publications

References 34 publications

A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

Optimization of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Schistosoma Mansoni (Trematoda: Digenea) Detection in Biomphalaria spp. from Endemic Areas for Schistosomiasis in Brazil

Simultaneous genotyping of snails and infecting trematode parasites using high‐throughput amplicon sequencing

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