Trypanosoma cruzi: parasite detection and strain discrimination in chronic chagasic patients from northeastern Brazil using PCR amplification of kinetoplast DNA and nonradioactive hybridization.
Blood samples from 172 individuals from northeastern Brazil were subjected to PCR amplification of Trypanosoma cruzi-specific kDNA sequences. This method enabled us to detect parasite DNA in 21 of 47 patients that were serologically positive. In addition, 1 patient that gave doubtful results with chagasic serology was confirmed as positive by PCR. We applied the same PCR detection method to the feces of wild triatomines captured in the same region, obtaining three positive results that were confirmed by microscopic examination. The 25 amplified products obtained in this study were then reamplified with primers that gave a final amplicon containing sequences from the most variable region of kDNA minicircles. These were used as probes in hybridization experiments aimed at defining the degree of relatedness between the strains infecting humans and insects based on kDNA homologies. We found that the amplification products from the three triatomines were related and showed no cross-hybridization with those obtained from human infections. Eight amplified products from human infections showed no cross-hybridization and did not hybridize with products from other patients. This indicates that the strains of T. cruzi circulating in the region present a high level of genetic heterogeneity. Finally, a number of amplified products hybridized with amplicons that did not hybridize with each other, indicating that infections with a parasite population presenting a mixed kDNA content (either due to different strains of T. cruzi or to a hybrid parasite) are a more frequent event than previously thought.
Department of Biochemistry and Molecular Biology, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.
C
C
Britto
M A
MA
Cardoso
C
C
Ravel
A
A
Santoro
J B
JB
Pereira
J R
JR
Coura
C M
CM
Morel
P
P
Wincker
D000818:Animals; D001938:Brazil / Q000453:epidemiology; D014355:Chagas Disease / Q000097:blood / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology; D002908:Chronic Disease; D004272:DNA, Mitochondrial / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000302:isolation & purification*; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology; D017720:Molecular Epidemiology; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012698:Serologic Tests; D014224:Triatoma / Q000469:parasitology; D014349:Trypanosoma cruzi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D004272:DNA, Mitochondrial; D016054:DNA, Protozoan