Cruzi Lineages Research Articles

Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding

Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes.

Sequencing and analysis of chromosomal extremities of Trypanosoma rangeli in comparison with Trypanosoma cruzi lineages

The aim of this study was to investigate the genetic variability of sequences present in the chromosome ends of Trypanosoma rangeli strains defined by the presence (+) or absence (-) of KP1 minicircles, and to compare the mean terminal restriction fragment (TRF) lengths to those of Trypanosoma cruzi populations representative of groups TcI, TcII, TcIV, and TcVI. Southern blots containing RsaI-digested genomic DNA of T. rangeli KP1(+) strains, T. rangeli KP1(-) strains, and T. cruzi strains were probed with the previously described subtelomeric sequences (170 bp) of T. rangeli and with telomeric hexamer repeats. Mean TRF length analysis showed that the chromosome ends of T. rangeli are distinctly organized, with TRFs ranging from 1.3 to 9 kb for KP1(+) strains and from 0.3 to 5.0 kb for KP1(-) strains. In T. cruzi, TRF length ranged from 0.2 to 9 kb and no association with the genotype of the parasite could be established. Sequence analysis of the 170-bp amplicons revealed the occurrence of sequence polymorphisms in the subtelomeric region between and within KP1(+) and KP1(-) strains. The GTT triplet was detected in all KP1(+) strains, except for strain Cas4, but not in any of the KP1(-) strains. The dendrogram constructed by alignment of all T. rangeli strains showed the division into two main groups, mainly related to the presence or absence of the KP1 minicircle. In conclusion, the present results extend the genotype differences demonstrated by kDNA and karyotype analysis in T. rangeli to the chromosome ends of the parasite.

Benznidazole alters the pattern of Cyclophosphamide-induced reactivation in experimental Trypanosoma cruzi-dependent lineage infection

The factors involved in the reactivation of chronic Chagas disease infection are not clear enough and may be related to host immune unbalance and/or parasite genetic diversity. To evaluate the role of the Trypanosoma cruzi genetic background in the Chagas disease reactivation, we inoculated Cyclophosphamide-immunosupressed (CyI) Swiss mice with clonal stocks from T. cruzi I (Cuica cl1, P209 cl1, Gamba cl1, SP104 cl1), T. cruzi II (IVV cl4, MVB cl8) and T. cruzi (Bug2148 cl1, MN cl2) lineages. We used the parasitemia as the parameter for Chagas disease reactivation and observed that CyI animals infected with T. cruzi stocks showed no reactivation and those infected with T. cruzi II stocks showed only 5% of reactivation. In contrast, immunosuppressed mice infected with stocks from T. cruzi I lineage showed 77.5 and 51.25% reactivation of the infection when Cyclophosphamide treatment was performed 60 and 180 days after inoculation, respectively. Next, we evaluated the efficacy of the Benznidazole (Bz) pre-treatment in reducing or preventing the recurrence of the infection in these CyI animals. In general, the percentage of the parasite recurrence was not altered among the CyI mice that received the Bz pre-treatment during the acute phase of the infection. Interestingly, when pre-Bz treatment was performed during the chronic phase, we observed two different patterns of response: (i) an increased protection among the animals inoculated with the SP104 cl1 (genotype 19) and Cuica cl1 (genotype 20) stocks; (ii) an increased percentage of parasitemia reactivation among mice inoculated with Gamba cl1 (genotype 19) and P209 cl1 (genotype 20) T. cruzi stocks. Our results corroborate our hypothesis by showing that the T. cruzi genetic background in combination with specific Bz treatment has an important role in the Chagas disease reactivation in immunosuppressed animals.

Trypanosoma cruzi IIc: Phylogenetic and Phylogeographic Insights from Sequence and Microsatellite Analysis and Potential Impact on Emergent Chagas Disease

Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.

The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania: looking back and to the future

Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e--now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.

Wild Ecotopes and Food Habits of Triatoma longipennis Infected by Trypanosoma cruzi Lineages I and II in Mexico

The control of wild triatomine populations that can invade dwellings is a major challenge for Chagas disease control in Mexico, but a better knowledge of the biology of these populations is required to develop appropriate control methods. We describe a new terrestrial ecotope of Triatoma longipennis, a principal vector in the occidental part of Mexico, in addition to its previously identified niche in rock pile boundary walls. Analysis of feeding hosts in the two ecotopes showed that this species is able to diversify its food sources outside of the principal hosts, Dasypus novemcinctus and Procyon lotor, and to disperse in search of new meals. Moreover, T. longipennis are strongly infected not only by the Trypanosoma cruzi I lineage found in the domestic cycle, but also by T. cruzi lineage II. The impact of T. cruzi II on human infection remains to be determined.

A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

Probing population dynamics of Trypanosoma cruzi during progression of the chronic phase in chagasic patients.

Our research aimed to characterize the genetic profiles of 102 Trypanosoma cruzi isolates recently obtained from 44 chronic chagasic patients from different regions of the states of Minas Gerais and Goiás in Brazil. At least two isolates were obtained from each patient at different times in order to study the parasite population dynamics during disease progression in the chronic phase. The isolates were characterized molecularly by genotyping the 3' region of the 24S alpha rRNA, the mitochondrial cytochrome oxidase subunit 2 (COII) gene, and the intergenic region of the spliced leader intergenic region (SL-IR) gene. Seventy-seven isolates were analyzed for nine microsatellite loci. The data presented here show a strong correlation between the T. cruzi lineage II (T. cruzi II) and human infection in these regions of Brazil. Interestingly, isolates from two patients were initially characterized (by rRNA genotyping) as T. cruzi I and hybrid strains, but subsequent analyses of the COII and SL-IR genes confirmed that those isolates belonged to T. cruzi III and a hybrid group, respectively. Our results confirm the risk of misclassifying T. cruzi isolates on the basis of analysis of a single molecular marker. The microsatellite profiles showed that different isolates obtained from the same patient were genetically identical and monoclonal. Exceptions were observed for T. cruzi isolates from two patients who presented differences for the SCLE11 locus and also from two other patients who showed amplification of three peaks for a microsatellite locus (TcAAAT6), implying that they were multiclonal. On the basis of the findings of the studies described here, we were not able to establish a correlation between the clinical forms of Chagas' disease and the genetic profiles of the T. cruzi isolates.

Phylogenetic analysis of microsatellite markers further supports the two hybridization events hypothesis as the origin of the Trypanosoma cruzi lineages

To better understand the evolution of the etiologic agent of Chagas disease, we cloned and sequenced 25 alleles from five Tripanosoma cruzi microsatellite markers. The study of the sequences showed highly conserved alleles present in T. cruzi clones belonging to TCI, TCIIc, and TCIIe. This result was also confirmed by the phylogenetic analysis of MCLE01 allele sequences. The examination by capillary electrophoresis of six microsatellite markers from 19 T. cruzi clones showed a high proportion of the alleles found both in the TCI and TCII sublineages. The phylogenetic reconstruction of these 19 clones produced a tree with two major clusters with bootstrap support of 100% and 95%. The first cluster includes T. cruzi clones belonging to the TCI and TCIIa lineages. The second cluster is composed of TCI, TCIIc, TCIId, and TCIIe T. cruzi clones. The analysis of five microsatellite markers in the CLBrener genome showed that almost all the microsatellite markers are synteny; non-Esmeraldo and Esmeraldo haplotypes probably come from the TCIIc and TCIIb lineages. Taken together, our results are in agreement with the two hybridization events hypothesis as the origin of current T. cruzi lineages.

Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosoma cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TCI presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TCI and TCIIa from wild primates (16 TCI and five TCIIa), Rhodnius spp. (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TCI and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T. cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region.

Molecular Identification of Trypanosoma cruzi I Tropism for Central Nervous System in Chagas Reactivation Due to AIDS

Trypanosoma cruzi lineages, microsatellite allelic polymorphism, and mithocondrial gene haplotypes were directly typified from peripheral blood and cerebrospinal fluid specimens of a Bolivian patient with Chagas disease with accompanying AIDS and central nervous system severe involvement. Of note, the patient's blood was infected by a mixture of T. cruzi I and T. cruzi IId/e polyclonal populations while the cerebrospinal fluid showed only a monoclonal T. cruzi I population. Our findings do not corroborate the original assumption of innocuity for T. cruzi I in the southern cone of the Americas and highlight lineage I tropism for central nervous system causing lethal Chagas reactivation.

Genotyping of Panamanian Trypanosoma cruzi stocks using the calmodulin 3′UTR polymorphisms

Mutations in the 3' untranslated region of calmodulin gene have recently been reported to be specific to different Trypanosoma cruzi lineages. In the present report, this molecular marker was used to genotype 24 T. cruzi isolates from humans and vectors from different endemic areas in Panama. The finding of solely T. cruzi I genotype might explain the low morbidity of Chagas' disease in the region when compared to other countries in Latin America.

Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the β-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

High variability of Colombian Trypanosoma cruzi lineage I stocks as revealed by low-stringency single primer-PCR minicircle signatures

In Colombia, high genetic variability has been found among Trypanosoma cruzi stocks isolated from different vector and host species, using isoenzyme analysis and RFLP of total kinetoplastid DNA (kDNA), suggesting that several genetically related T. cruzi populations might be present within a single geographical area or adjacent ones. The objective of this study was to use the low-stringency single primer (LSSP)-PCR technique on variable regions of kDNA minicircles of T. cruzi to determine possible genetic relationships among stocks from distinct geographical regions of Colombia and different vector species and hosts. Although LSSP-PCR analysis showed a high genetic variability among 30 Colombian T. cruzi stocks, 29 of them belonged to T. cruzi lineage I, confirming that this lineage is predominant in different vector and host species from Colombia. Interestingly, one stock isolated from a Pastrongylus geniculatus bug was identified as T. cruzi lineage IIb, using PCR strategies targeted to the intergenic region of miniexon genes, a sequence encoding the D7 domain of the 24sα ribosomal genes and the A10 fragment, being this finding, the first description of this lineage in Colombia. The LSSP-PCR signatures allowed correlation of most isolates with their respective geographical origins, and in one case from host and vector specimens at a same region, suggesting a transmission event. Moreover, variations in LSSP-PCR profiles among T. cruzi I stocks from a same region suggest that they may have a multiclonal character. Our results show that LSSP-PCR is a fast, valuable technique for characterization of intra-lineage polymorphism among T. cruzi stocks.

CONGENITAL CHAGAS DISEASE IN BOLIVIA IS NOT ASSOCIATED WITH DNA POLYMORPHISM OF TRYPANOSOMA CRUZI

This study aims to typify the Trypanosoma cruzi (sub)lineage(s) in umbilical cord blood of congenitally infected Bolivian newborns, using PCR amplifications of "Region Markers", mini-exon or kDNA fragments followed by hybridization or sequencing. New probes were also designed to distinguish three variants within the TcIId sublineage. The IIb, IId, or IIe T. cruzi sublineages, as well as different variants of the IId sublineage, were detected in infected neonates, whereas mixed infections were not found. The frequencies of the IId sublineage were similar in neonates (95.1%) and adults of the same area (94.1%). The IId-infected newborns displayed either asymptomatic, or severe and fatal clinical forms of congenital Chagas disease, as well as low or high parasitemia. Altogether these data show that T. cruzi DNA polymorphism, based on the presently available markers, is not associated with the occurrence of congenital infection or the development of severe clinical forms of congenital Chagas disease.

Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium

The isolation of biological clones of Trypanosoma cruzi by microscopically dispensing individual organisms or by serial dilution is laborious and time consuming. The inability to resolve mixed T. cruzi infections, from vectors and hosts, and to isolate clones of slow growing genotypes by efficient plating on solid media, has hindered characterisation studies and downstream applications. We have devised and validated a sensitive, solid medium plating technique for rapid in vitro isolation of clones representative of all the recognised T. cruzi lineages (TCI, TCIIa–e), including the slow growing strain CANIII (TC IIa) and Trypanosoma rangeli, with high plating efficiencies. Furthermore, the method is effective for the isolation of clones directly from silvatic triatomine bugs and from experimentally infected mice harbouring mixed infections, allowing resolution of multiclonal infections from varied sources.

Long-term reduction of Trypanosoma cruzi infection in sylvatic mammals following deforestation and sustained vector surveillance in northwestern Argentina

Long-term variations in the dynamics and intensity of sylvatic transmission of Trypanosoma cruzi were investigated around eight rural villages in the semiarid Argentine Chaco in 2002–2004 and compared to data collected locally in 1984–1991. Of 501 wild mammals from 13 identified species examined by xenodiagnosis, only 3 (7.9%) of 38 Didelphis albiventris opossums and 1 (1.1%) of 91 Conepatus chinga skunks were infected by T. cruzi. The period prevalence in opossums was four-fold lower in 2002–2004 than in 1984–1991 (32–36%). The infection prevalence of skunks also decreased five-fold from 4.1–5.6% in 1984–1991 to 1.1% in 2002–2004. Infection in opossums increased with age and from summer to spring in both study periods. The force of infection per 100 opossum-months after weaning declined more than six-fold from 8.2 in 1988–1991 to 1.2 in 2002–2004. Opossums were mainly infected by T. cruzi lineage I and secondarily by lineage IId in 1984–1991, and only by T. cruzi I in 2002–2004; skunks were infected by T. cruzi IId in 1984–1991 and by IIc in 2002–2004. The striking decline of T. cruzi infection in opossums and skunks occurred in parallel to community-wide insecticide spraying followed by selective sprays leading to very low densities of infected Triatoma infestans in domestic and peridomestic habitats since 1992; to massive deforestation around one of the villages or selective extraction of older trees, and apparent reductions in opossum abundance jointly with increases in foxes and skunks. These factors may underlie the dramatic decrease of T. cruzi infection in wild reservoir hosts.

Trypanosoma cruzi: Variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.

Comparative analysis of genomic sequences suggests that Trypanosoma cruzi CL Brener contains two sets of non-intercalated repeats of satellite DNA that correspond to T. cruzi I and T. cruzi II types

Approximately 10% of the Trypanosoma cruzi genome is formed by a satellite DNA, composed by 195-bp repeats organized in 30 ± 10 kb clusters in some, but not all chromosomes. Here, the satellite DNA of six representative T. cruzi strains was sequenced and used for phylogenetic inference. The results show that CL Brener contains satellite repeats from T. cruzi I and T. cruzi II strains, although type II sequences are more abundant. The presence of types I and II sequences extends previous propositions that genetic exchange between the two major T. cruzi lineages have occurred in CL Brener, although our data accommodate alternative scenarios of hybridization within T. cruzi II, as proposed by others. Altogether, present data suggest a complex origin for CL Brener. Sequence analysis of satellites isolated from chromosomal bands indicates that satellite DNA sequences are not chromosome specific. Neighbor analysis of in tandem satellite DNAs containing up to five repeats shows that each cluster contains only one type of sequence. Consequently, clusters with intercalated types I and II repeats were not found. We propose that the CL Brener genome contains large pieces of satellite DNA originated mainly from chromosomes of T. cruzi II with introgression of T. cruzi I lineage.

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Sα rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa–IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Sα rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Sα rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Sα rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Sα rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.