TY - JOUR
T1 - Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites
AU - Valadares, Helder Magno Silva
AU - Pimenta, Juliana Ramos
AU - de Freitas, Jorge Marcelo
AU - Duffy, Tomás
AU - Bartholomeu, Daniella C.
AU - de Paula Oliveira, Riva
AU - Chiari, Egler
AU - Moreira, Maria da Consolação Vieira
AU - Filho, Geraldo Brasileiro
AU - Schijman, Alejandro Gabriel
AU - Franco, Glória Regina
AU - Machado, Carlos Renato
AU - Pena, Sérgio Danilo Junho
AU - Macedo, Andréa Mara
N1 - Funding Information:
This work was supported by PRONEX CNPq/FAPEMIG, CAPES, WHO. We thank the medicine students, Laura Gomide, Aline V. Santana, Andrea F. Silveira and Henrique C. R. Galvão, for helping in the experiments, Neuza A. Rodrigues and Kátia B. Gonçalves for expert technical assistance and to Drs Hector Freilij, Mirta Diez and M. Elena Seidenstein for clinical diagnosis and follow-up of Argentinean Chagas disease patients. Partial support of PIP 5369 and PICT 33955 to A.G.S.
PY - 2008/6
Y1 - 2008/6
N2 - 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.
AB - 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.
KW - Chagas disease
KW - Full nested PCR
KW - Genome project
KW - Polymorphic microsatellites
KW - Trypanosoma cruzi
UR - http://www.scopus.com/inward/record.url?scp=41949140657&partnerID=8YFLogxK
U2 - 10.1016/j.ijpara.2007.10.017
DO - 10.1016/j.ijpara.2007.10.017
M3 - Artículo Científico
C2 - 18154957
AN - SCOPUS:41949140657
SN - 0020-7519
VL - 38
SP - 839
EP - 850
JO - International Journal for Parasitology
JF - International Journal for Parasitology
IS - 7
ER -