title: Deep sequencing of the Trypanosoma cruzi GP63 surface proteases reveals diversity and diversifying selection among chronic and congenital Chagas disease patients.
abstract: BACKGROUND
  Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology.
  
  
  RESULTS
  A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus.
  
  
  CONCLUSIONS
  Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.
authors: The London School of Hygiene and Tropical Medicine, London, United Kingdom; Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, University of Wales, Bangor, Bangor, Gwynedd, United Kingdom.
authors: Martin S
authors: MS
authors: Llewellyn
authors: The London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors: Louisa A
authors: LA
authors: Messenger
authors: Laboratório de Pesquisa da doença de Chagas, Hospital das Clínicas da Universidade Federal de Goiás, Brazil.
authors: Alejandro O
authors: AO
authors: Luquetti
authors: Facultad de Medicine, Universidad Mayor de San Simon, Cochabamba, Bolivia.
authors: Lineth
authors: L
authors: Garcia
authors: Facultad de Medicine, Universidad Mayor de San Simon, Cochabamba, Bolivia.
authors: Faustino
authors: F
authors: Torrico
authors: Laboratório de Pesquisa da doença de Chagas, Hospital das Clínicas da Universidade Federal de Goiás, Brazil.
authors: Suelene B N
authors: SB
authors: Tavares
authors: Institut de Biologie Integrative et de Systemes, Universite de Laval, Quebec, Canada.
authors: Bachar
authors: B
authors: Cheaib
authors: Institut de Biologie Integrative et de Systemes, Universite de Laval, Quebec, Canada.
authors: Nicolas
authors: N
authors: Derome
authors: Centre National de Génotypage, CEA, Evry, Paris, France.
authors: Marc
authors: M
authors: Delepine
authors: Centre National de Génotypage, CEA, Evry, Paris, France.
authors: Céline
authors: C
authors: Baulard
authors: Centre National de Génotypage, CEA, Evry, Paris, France.
authors: Jean-Francois
authors: JF
authors: Deleuze
authors: Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors: Sascha
authors: S
authors: Sauer
authors: The London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors: Michael A
authors: MA
authors: Miles
mesh_terms: D000818:Animals; D001838:Bolivia; D001938:Brazil; D014355:Chagas Disease / Q000151:congenital / Q000469:parasitology*; D002908:Chronic Disease; D015331:Cohort Studies; D005260:Female; D005786:Gene Expression Regulation; D014644:Genetic Variation; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D018445:Infectious Disease Transmission, Vertical; D008297:Male; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D010447:Peptide Hydrolases