Development of an Amplicon-Based Next-Generation Sequencing Protocol to Identify
            <i>Leishmania</i>
            Species and Other Trypanosomatids in Leishmaniasis Endemic Areas

Patiño, Luz Helena; Castillo-Castañeda, Adriana; Muñoz, Marina; Rojas‐Jaimes, Jesús; Luna-Niño, Nicolas; Hernández, Carolina; Ayala, Martha S.; Fuya, Patricia; Méndez, Claudia; Hernández‐Pereira, Carlos E.; Delgado, Lourdes; Sandoval-Ramírez, Claudia M.; Urbano, Plutarco; Paniz‐Mondolfi, Alberto; Ramírez, Juan David

doi:10.1128/spectrum.00652-21

Cited by 14 publications

(14 citation statements)

References 71 publications

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“…NGS allowed the identification of a broad range of samples simultaneously, in addition to identifying the possible parasite species circulating in a given sample, even using culture isolate samples, which have gone through parasite species selection process, which could affect the results observed. Some studies have already used this methodology to identify trypanosomatids in samples from different mammal species, and it was possible to identify even more than three species in a single sample and even species of the Kinetoplastea class infecting mammals ( Barbosa et al., 2017 ; Dario et al., 2017a ; Dario et al., 2017b ; Cooper et al., 2018 ; Huggins et al., 2019 ; Patiño et al., 2021 ). This is a promising methodology in parasitological studies that is able to unravel a world that we did not have access to before due to diagnostic limitations.…”

Section: Discussionmentioning

confidence: 99%

“…As the one-parasite-one-disease concept generally predominates, the focus has always been on T. cruzi or Leishmania sp., and the possible coinfecting trypanosomatids have been relegated to the background. Mixed infections between different trypanosomatid species and genotypes are very common in nature ( Barbosa et al., 2017 ; Dario et al., 2017a ; Jansen et al., 2018 ; Patiño et al., 2021 ). The determination of mixed infections is essential for the understanding of parasitological events, especially the resulting interactions between the parasite and its hosts.…”

Section: Introductionmentioning

confidence: 99%

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Trypanosomatid Richness Among Rats, Opossums, and Dogs in the Caatinga Biome, Northeast Brazil, a Former Endemic Area of Chagas Disease

Dario

Furtado

Lisboa

et al. 2022

Front. Cell. Infect. Microbiol.

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Parasites are important components of the immense n-dimensional trophic network that connects all living beings because they, among others, forge biodiversity and deeply influence ecological evolution and host behavior. In this sense, the influence of Trypanosomatidae remains unknown. The aim of this study was to determine trypanosomatid infection and richness in rats, opossums, and dogs in the semiarid Caatinga biome. We submitted DNA samples from trypanosomatids obtained through axenic cultures of the blood of these mammals to mini exon multiplex-PCR, Sanger, and next-generation sequencing targeting the 18S rDNA gene. Phylogenetic analyses were performed to identify genetic diversity in the Trypanosomatidae family. Shannon, Simpson, equability, and beta-diversity indices were calculated per location and per mammalian host. Dogs were surveyed for trypanosomatid infection through hemocultures and serological assays. The examined mammal species of this area of the Caatinga biome exhibited an enormous trypanosomatid species/genotypes richness. Ten denoised Operational Taxonomic Units (ZOTUs), including three species (Trypanosoma cruzi, Trypanosoma rangeli and Crithidia mellificae) and one Trypanosoma sp. five genotypes/lineages (T. cruzi DTU TcI, TcII, and TcIV; T. rangeli A and B) and four DTU TcI haplotypes (ZOTU1, ZOTU2, ZOTU5, and ZOTU10 merged), as well as 13 Amplicon Sequence Variants (ASVs), including five species (T. cruzi, T. rangeli, C. mellificae, Trypanosoma dionisii, and Trypanosoma lainsoni), five genotypes/lineages (same as the ZOTUs) and six DTU TcI haplotypes (ASV, ASV1, ASV2, ASV3, ASV5 and ASV13), were identified in single and mixed infections. We observed that trypanosomatids present a broad host spectrum given that species related to a single host are found in other mammals from different taxa. Concomitant infections between trypanosomatids and new host-parasite relationships have been reported, and this immense diversity in mammals raised questions, such as how this can influence the course of the infection in these animals and its transmissibility. Dogs demonstrated a high infection rate by T. cruzi as observed by positive serological results (92% in 2005 and 76% in 2007). The absence of positive parasitological tests confirmed their poor infectivity potential but their importance as sentinel hosts of T. cruzi transmission.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Trypanosomatid Richness Among Rats, Opossums, and Dogs in the Caatinga Biome, Northeast Brazil, a Former Endemic Area of Chagas Disease

Dario

Furtado

Lisboa

et al. 2022

Front. Cell. Infect. Microbiol.

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“…We selected three serum and three bone marrow aspirates smears from five patients with parasitological or serological diagnosis of VL (Table 1). DNA extracted from each sample was used for Leishmania species identification by Sanger sequencing and HSP70amplicon-based-NGS as per protocol (Patiño et al, 2021). Leishmania spp.…”

Section: Graphical Abstractmentioning

confidence: 99%

Amplicon-based next-generation sequencing reveals the co-existence of multiple Leishmania species in patients with visceral leishmaniasis

Castillo-Castañeda

Patiño

Muñoz

et al. 2022

International Journal of Infectious Diseases

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…Another study used high-resolution melt (HRM) qPCR to detect Leishmania [ 33 ]. Hitherto, only a handful of studies used NGS technology in the identification of Leishmania that used ITS1 and HSP70 (heath shock protein) with the former using longer targets (500 bp) and ignoring validity checks, thus reducing sensitivity and specificity [ 36 , 62 , 63 ].…”

Section: Discussionmentioning

confidence: 99%

Concurrent molecular characterization of sand flies and Leishmania parasites by amplicon-based next-generation sequencing

et al. 2022

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Background Phlebotomine sand flies are vectors of Leishmania parasites, which are the causative agents of leishmaniasis. Herein, we developed an amplicon-based next-generation sequencing (Amp-NGS) to characterize sand flies and Leishmania parasites simultaneously targeting partial fragments of 18S rDNA and ITS1 genes, respectively. Methods Our assay was optimized using reference sand fly (n = 8) and Leishmania spp. (n = 9) samples and validated using wild-caught sand flies from Palestine. The assay was highly specific, and all DNA references were successfully identified to the species level. Results Among the wild-caught sand flies (n = 187), Phlebotomus spp. represented 95% of the collected samples (177/187), including Ph. sergenti (147/187, 79%), Ph. papatasi (19/187, 10.2%), Ph. perfiliewi (3/187, 1.6%), Ph. tobbi (2/187, 1.2%) and Ph. syriacus (6/187, 3.2%). Sergentomyia spp. represented only 5% (10/187) of the collected samples and included S. dentata (n = 6), S. fallax (n = 2), S. schwetzi (n = 1) and S. ghesquiere (n = 1). The study observed strong positive correlation between sand fly identification results of the Amp-NGS and morphological identification method (r = 0.84, df = 185, P < 0.001). Some discrepancies between the two methods in the identification of closely related species (i.e. Ph. perfiliewi, Ph. tobbi and Ph. syriacus) were observed. Leishmania DNA was detected and identified as L. tropica in 14 samples (14/187, 7.5%). Conclusions Our assay was sensitive to detect (limit of detection was 0.0016 ng/reaction) and identify Leishmania DNA in sand flies, thus representing a new tool for studying sand flies and their associated Leishmania parasites in endemic areas. Graphical Abstract

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Development of an Amplicon-Based Next-Generation Sequencing Protocol to Identify Leishmania Species and Other Trypanosomatids in Leishmaniasis Endemic Areas

Cited by 14 publications

References 71 publications

Trypanosomatid Richness Among Rats, Opossums, and Dogs in the Caatinga Biome, Northeast Brazil, a Former Endemic Area of Chagas Disease

Trypanosomatid Richness Among Rats, Opossums, and Dogs in the Caatinga Biome, Northeast Brazil, a Former Endemic Area of Chagas Disease

Amplicon-based next-generation sequencing reveals the co-existence of multiple Leishmania species in patients with visceral leishmaniasis

Concurrent molecular characterization of sand flies and Leishmania parasites by amplicon-based next-generation sequencing

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