Computational protocol: Spatial and Molecular Epidemiology of Giardia intestinalis Deep in the Amazon, Brazil

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Protocol publication

[…] This study was a cross-sectional survey performed in 433 children from Santa Isabel do Rio Negro in 2011 (). This small city in Brazilian Amazon was occupied mainly by Amerindians, descendent from the Tukano and Aruak speaking societies. Although the overall population of this area was approximately 18,000 people, this study was conducted in the urban area, comprised of approximately 5,000 inhabitants, distributed among six districts: Aparecida (APA), Centro (CEN), Santa Inês (SI), São José Operário (SJO), São Judas Tadeu (SJT), and Santana (SAN). All children included in our study were at maximum 14 years old. None of them presented with diarrhea during the study. Containers without preservatives were distributed for stool samples collection, and parasitological tests were performed using ether sedimentation technique []Georeferencing was performed with a Global Position System in the SAD-69 geodetic datum. Spatial data were analyzed in a GIS platform using ArcGis 9.3® software (Environmental Systems Research Institute, Redlands, CA-USA). Maps were generated using the kernel density estimation method, and only first order effects were evaluated. The maps were made using data provided by OpenStreetMap® available under the Open Database License (https://www.openstreetmap.org/copyright).DNA was extracted, in a field laboratory, only from parasitologicaly confirmed G. intestinalis-positive stool samples using the ZR Fungal/Bacterial DNA kit (ZymoResearch, Irvine-USA). For the amplification of the 753-bp β-giardin (βG) gene fragment we utilized the G7-G759 primers, as described by Cacciò et al. []. Products were purified using the Illustra-GFX kit (GE Healthcare, Pittsburgh, PA-USA) and sequenced with the ABI-BigDye Terminator kit (Applied Biosystems, Foster City, CA-USA) using ABI 3730 (Applied Biosystems) automated sequencer. In addition, sequences that presented double peaks were cloned using pGEM® T-Easy (Promega, Madison, WI-USA). Briefly, the inserts were amplified by PCR using the M13 primer and sequenced []. We used the Bioedit-7.1 and Mega-6.0 in order to edit and align the sequences.Bayesian and maximum-likelihood phylogenetic trees based on 657-bp βG sequences were inferred with BEAST-1.8 and PhyML-3.0, respectively. The Akaike and Bayesian Information Criteria of jMODELTEST-2 were used to elect Tamura-Nei with four gamma categories as the best-fit evolutionary model for the dataset. Eighteen orthologous sequences representing the diversity of G. intestinalis (six of the eight known assemblages) were retrieved from GenBank and added to the analyses. Genealogies were reconstructed with Network-4.6 (Fluxus-Engineering, Inc.) using the median-joining method with maximum-parsimony post-processing. […]

Pipeline specifications

Software tools BioEdit, MEGA, BEAST, PhyML, jModelTest
Application Phylogenetics
Organisms Giardia intestinalis, Homo sapiens
Diseases Infection, Pneumatosis Cystoides Intestinalis