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PublicationDevelopment of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection(Elsevier, 2014-04-01)
;Nzelu C.O. ;Gomez E.A. ;Sakurai T. ;Martini-Robles L. ;Uezato H. ;Mimori T. ;Katakura K. ;Hashiguchi Y.Kato H.Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1. h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)-mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries. © 2013 Elsevier B.V. -
PublicationOccurrence of Giardia intestinalis and Cryptosporidium sp. in wastewater samples from São Paulo State, Brazil, and Lima, Peru(Springer Verlag, 2016-11-01)
;Aguiar B. ;Sato M.I.Z. ;Guerrero J.A. ;Hachich E. ;Matté G.R. ;Dropa M. ;Matté M.H.de Araújo R.S.The objectives of the study were to detect and genotype Cryptosporidium spp. and Giardia intestinalis in wastewater samples obtained from five cities with high transit of people in the State of São Paulo, Brazil, and at the entrance of a Wastewater Treatment Plant (WWTP) in Lima, Peru. Samples were collected and concentrated by centrifugation. The genomic DNA was extracted for molecular characterization by nested PCR for Cryptosporidium and double nested PCR for Giardia, followed by sequencing and phylogenetic analysis. G. intestinalis was found in 63.6 % of the samples, and the human assemblages A and B were identified. Cryptosporidium sp. was found in 36.4 % of the samples, and the species were corresponding to Cryptosporidium hominis, Cryptosporidium cuniculus, and Cryptosporidium muris. Results revealed the presence of human pathogenic Cryptosporidium species and G. intestinalis human pathogenic assemblages. Molecular tools highlight the importance to map the genetic diversity of these parasites, as well as to detect their epidemiological circulation pathway in the environment. -
PublicationPhylogenetic relationships of Strongyloides species in carnivore hosts(Elsevier Ireland Ltd, 2020-10-01)
;Ko P.P. ;Suzuki K. ;Aung M.P.P.T.H.H. ;Htike W.W. ;Yoshida A. ;Morishita K. ;Maruyama H.Nagayasu E.Strongyloides stercoralis is a parasitic nematode and a major pathogen responsible for human strongyloidiasis. The presence of this species in the dog population has led to an interest in studying the phylogenetic relationships among Strongyloides spp. in carnivore hosts. In the present study, Strongyloides spp. from various carnivore hosts (raccoon, Japanese badger, Siberian weasel, raccoon dog, masked palm civet, and domestic cat) were sought. Except for civets, Strongyloides spp. were identified in all host species. Based on 18S rDNA sequences, nine OTUs (operational taxonomy units) were identified. Molecular phylogenetic analyses using 18S[sbnd]28S rDNA and mitochondrial cox1 (cytochrome c oxidase subunit 1) sequences clustered them into two groups. The first group (named the stercoralis/procyonis group) was comprised of six OTUs and occurred in cats, raccoon dogs, raccoons (S. procyonis), Siberian weasels, and Japanese badgers and included S. stercoralis from humans and dogs. The second group (named the planiceps group) was made up of Strongyloides spp. from raccoon dogs (two OTUs) and one OTU from Siberian weasels. Subsequent analysis using almost the full-length nucleotide sequences of protein-coding genes in their mitochondrial genomes placed Strongyloides spp. of cats in a sister taxon position to S. stercoralis, whereas S. procyonis from raccoons was more distantly related to them. The presence of Strongyloides spp. from various carnivore hosts, which are close relatives of S. stercoralis, suggests this group of Strongyloides (the stercoralis/procyonis group) essentially evolved as parasites of carnivores, although more data on Strongyloides spp. from primate hosts are needed.