title: Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.
abstract: The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci--six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, Brazil.
authors.forename: Helder Magno Silva
authors.initials: HM
authors.lastname: Valadares
authors.forename: Juliana Ramos
authors.initials: JR
authors.lastname: Pimenta
authors.forename: Jorge Marcelo
authors.initials: JM
authors.lastname: de Freitas
authors.forename: Tomás
authors.initials: T
authors.lastname: Duffy
authors.forename: Daniella C
authors.initials: DC
authors.lastname: Bartholomeu
authors.forename: Riva de Paula
authors.initials: Rde P
authors.lastname: Oliveira
authors.forename: Egler
authors.initials: E
authors.lastname: Chiari
authors.forename: Maria da Consolação Vieira
authors.initials: Mda C
authors.lastname: Moreira
authors.forename: Geraldo Brasileiro
authors.initials: GB
authors.lastname: Filho
authors.forename: Alejandro Gabriel
authors.initials: AG
authors.lastname: Schijman
authors.forename: Glória Regina
authors.initials: GR
authors.lastname: Franco
authors.forename: Carlos Renato
authors.initials: CR
authors.lastname: Machado
authors.forename: Sérgio Danilo Junho
authors.initials: SD
authors.lastname: Pena
authors.forename: Andréa Mara
authors.initials: AM
authors.lastname: Macedo
mesh_terms: D000818:Animals; D014355:Chagas Disease / Q000235:genetics* / Q000469:parasitology; D002874:Chromosome Mapping; D002908:Chronic Disease; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005787:Gene Frequency; D017125:Genes, Protozoan; D006321:Heart / Q000469:parasitology; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018895:Microsatellite Repeats; D018512:Parasitemia; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011247:Pregnancy; D012007:Rectum / Q000469:parasitology; D016415:Sequence Alignment; D012867:Skin / Q000469:parasitology; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers