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Determination of T. cruzi lineages analyzing minicircles DNA sequences

T. cruzi undergoes essentially clonal evolution with only very rare sexual recombination as described by Tibayrenc et al.24. The occurrence of hybridization in natural populations of T. cruzi has been unequivocally demonstrated.14,25,26 Nevertheless, the rarity of such sexual recombination allows propagation of clonal genotypes over long periods of time.26 Consequently, lineages of this parasite are identifiable on the basis of their genotype and phenotype. Recently, the nomenclature of T. cruzi intraspecific variability has been revised, taking into account the six principal



The identification of the lineage could be based either on genomic DNA sequences, on the polymorphism of isoenzymes (i.e., products of genomic DNA) or, on the presence of specific sequences in hypervariable kDNA region. It is largely accepted that at least two hybridization events occurred in T. cruzi natural populations in which a fusion between ancestral TC I—II (former DTU I and DTU Ilb, respectively) genotypes gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of TC III—TC IV (former DTU IIa and DTU IIc, respectively). The second hybridization was between TC II and TC III (former DTU IIb and DTU IIc, respectively) strains that generated TC V and TC VI (former DTUs IId and DTU IIe, respectively). It is noteworthy that the reference strain CL Brener, whose genome was first sequenced, pertains to a hybrid TC VI (former DTU IIe) lineage.

For various reasons developed later, the genetic pressure to maintain the constant gRNA encoding domain of minicircles appears to be low. It is therefore not surprising that the long and separate evolution of parasites led to the evolution of the sequence diversity of hypervariable domain minicircles. The relationships between nuclear genotypes specific to given lineages and sets of minicircles sequences is, however, maintained. Consequently, each lineage may be characterized by a set of minicircle sequences roughly specific to this lineage. The sequence diversity of minicircles is the foundation of various typing methods determining parasite lineage.

A first approach to T. cruzi kDNA characterization, described by Mattei et al.28 was based on the variability of restriction fragment length polymorphism (RFLP) of the minicircles. The kDNA should be purified, which requires extraction of kDNA from a large number of cultured parasites. Later, Morel et al.,29 used this method for genotyping T. cruzi strains and proposed the term “schizodeme” to refer to groups of parasites presenting the same pattern of kDNA.

Schizodeme typing is based on the separation of fragments of the digested kDNA by electrophoresis. Most of the digestions for schizodeme analysis were performed in polyacrylamide gels. When agarose gels were used, the resolution was not sufficient, and hybridization (southern blot) was necessary. These methods are time-consuming.

PCR amplification of hypervariable domain of minicircles was already described in 1989 by Sturm et al.30 Hypervariable domains are amplified using primers based on the constant region. An amplicon of approximately 320 bp was obtained. This amplicon contains the hypervariable domain but also about 100 bp of the constant domain (primers plus 60 bp of relatively constant sequence).

This PCR approach yielded good results with relatively pure parasites isolated from bugs, but did not allow direct identification of parasites in blood samples. In order to increase sensitivity and specificity, hybridization of amplicons was pro- posed.32 This approach was developed by Veas et al.,31 who amplified the kDNA hypervariable domain using modified primer containing restriction sites, allowing subsequent elimination of constant domains from amplicons (HVRm).

Probes prepared by Veas et al.31 were used for strain typing. Breniere et al. demonstrated that probes obtained by amplification of kDNA from clonet 39, hybridized only with DNA from clonet 39, probes obtained from clonet 43, hybridized only with DNA of clonet 43, and probes from clonet 20, hybridized with clonet 19 and 20, respectively32,33 Subsequently, the method was validated for epidemiological purposes, using total kDNA as probes.34 More recently it was used for typification of the Chile T. cruzi isolates.35

Systematic sequencing of large numbers of hypervariable sequences originating from different lineages confirmed sequence specificity of each group and demonstrated that if some hypervariable sequences are rare or unique, other sequences are frequently repeated in one lineage but, in proportion, varying from one strain to another. This was observed mainly in lineage TC V (former DTU IId).36

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