Ximena Coronado

Multilocus Polymerase Chain Reaction Restriction Fragment—Length Polymorphism Genotyping of Trypanosoma cruzi (Chagas Disease): Taxonomic and Clinical Applications

BACKGROUND Trypanosoma cruzi, the agent of Chagas disease, is subdivided into 6 discrete typing units (DTUs); their identification is important to understand clinical pleomorphism and track sylvatic DTUs that might (re-)invade domestic foci of the disease and jeopardize the running control programs. METHODS The genetic polymorphism of 12 loci was analyzed by multilocus polymerase chain reaction restriction fragment--length polymorphism (PCR-RFLP) analysis (MLP analysis) in a sample representative of the diversity within T. cruzi. We paid particular attention to genes involved in host-parasite relationships, because these may be prone to polymorphism as an adaptive answer to the immune selective pressure. RESULTS The results of MLP analysis were shown to agree with the current multilocus enzyme electrophoresis- and random amplified polymorphic DNA-based classification of T. cruzi in 6 DTUs, thereby providing a taxonomic validation of our method. Our data supported hypotheses of genetic recombination within T. cruzi. We demonstrated direct applicability of PCR-RFLP analysis to blood of mammal hosts and intestine content of vector insects. Domestic DTUs were encountered in wild animals, and, reciprocally, sylvatic DTUs were encountered in humans, raising questions about changes of transmission patterns. CONCLUSIONS MLP analysis represents a new alternative to existing molecular methods for T. cruzi typing. It might offer an invaluable support to clinical and epidemiological studies and to control programs.

T. cruzi OligoC-TesT: a simplified and standardized polymerase chain reaction format for diagnosis of Chagas disease.

Background PCR has evolved into one of the most promising tools for T. cruzi detection in the diagnosis and control of Chagas disease. However, general use of the technique is hampered by its complexity and the lack of standardization. Methodology We here present the development and phase I evaluation of the T. cruzi OligoC-TesT, a simple and standardized dipstick format for detection of PCR amplified T. cruzi DNA. The specificity and sensitivity of the assay were evaluated on blood samples from 60 Chagas non-endemic and 48 endemic control persons and on biological samples from 33 patients, 7 reservoir animals, and 14 vectors collected in Chile. Principal Findings The lower detection limits of the T. cruzi OligoC-TesT were 1 pg and 1 to 10 fg of DNA from T. cruzi lineage I and II, respectively. The test showed a specificity of 100% (95% confidence interval [CI]: 96.6%–100%) on the control samples and a sensitivity of 93.9% (95% CI: 80.4%–98.3%), 100% (95% CI: 64.6%–100%), and 100% (95% CI: 78.5%–100%) on the human, rodent, and vector samples, respectively. Conclusions The T. cruzi OligoC-TesT showed high sensitivity and specificity on a diverse panel of biological samples. The new tool is an important step towards simplified and standardized molecular diagnosis of Chagas disease.

Evolutionary history of Trypanosoma cruzi according to antigen genes

Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.

Molecular Epidemiology of Chagas Disease in the Wild Transmission Cycle: The Evaluation in the Sylvatic Vector Mepraia spinolai from an Endemic Area of Chile

The sylvatic transmission cycle of Chagas disease in Chile is composed of wild mammals and insects of the genus Mepraia. We determined infection rates and Trypanosoma cruzi genotypes in Mepraia spinolai. We collected 227 insects from two ecologically contrasting areas to assess T. cruzi infection. Polymerase chain reaction (PCR)-amplified minicircle DNAs were characterized by Southern blot and hybridization tests with genotype-specific probes. Infection in insects from the more fertile area was almost 2-fold higher than in the poorer area. The genotype TCI was the most prevalent and other genotypes such as TCIIb, TCIId, and TCIIe were found at lower rates. The areas differed in their genotype distribution but not in their genotype diversity. We suggest that the difference in abundance and richness of mammals between the areas may be producing the detected infection levels in vectors. Our results are compared with those reported for mammals from the same area.

Predominance of Trypanosoma cruzi genotypes in two reservoirs infected by sylvatic Triatoma infestans of an endemic area of Chile

We report results of Trypanosoma cruzi infection and parasite genotypes in the wild Octodon degus and synantropic reservoir Rattus rattus from an endemic area with sylvatic Triatoma infestans as the only detected vector. The infection status was determined by hemi-nested PCR directed to minicircles DNA and genotyping by hybridization tests with a panel of five specific probes, including two probes for TcI subgroups (clones 19 and 20). O. degus was found infected with 13.3% and mainly with sublineage TcIId, and less with TcIIb and TcI. Meantime the synantropic R. rattus was found infected with 27.7% and mainly with TcI and much less with TcIId, TcIIb and TcIIe. The results are discussed to explain the distribution of T. cruzi genotypes between these two reservoirs and the importance of sylvatic foci of T. infestans allowing the permanence of the wild and peridomestic cycle of Chagas disease.

Trypanosoma cruzi Genotypes of Insect Vectors and Patients with Chagas of Chile Studied by Means of Cytochrome b Gene Sequencing, Minicircle Hybridization, and Nuclear Gene Polymorphisms

Fifty-six Trypanosoma cruzi stocks from Chile and neighboring countries and different hosts, humans, and Triatoma infestans and Mepraia sp., vectors of domiciliary and natural environments were characterized by using three molecular markers. These were cytochrome b (Cyt b) gene sequencing, minicircle DNA blotting, and hybridization with five genotype-specific DNA probes and nuclear analysis of 1f8 and gp72 by polymerase chain reaction-restriction fragment length polymorphism. The results with all three molecular markers are concordant, with minor limitations, grouping T. cruzi stocks into four discrete typing units (DTUs) (TcI, TcII, TcV, and TcVI). TcI and TcII stocks were heterogeneous. TcI and TcII stocks were clustered in two main subgroups determined by Cyt b gene sequencing and minicircle hybridization. However, TcV and TcVI stocks were homogeneous and not differentiated by Cyt b gene sequencing or minicircle DNA hybridization. The discriminatory power and limitations of the molecular markers are discussed, as well as the distribution of the four DTUs in the domiciliary and sylvatic transmission cycles of Chile and the limitations of each marker for molecular epidemiological studies performed with T. cruzi stocks rather than the analysis of direct T. cruzi samples from natural hosts.

Differential distribution of Trypanosoma cruzi clones in human chronic chagasic cardiopathic and non-cardiopathic individuals.

PCR and Southern blot hybridization were used to determine the distribution of Trypanosoma cruzi clones in 37 chronic chagasic cardiopathic and non-cardiopathic patients. Parasite DNA amplified from peripheral blood or dejections of Triatoma infestans fed on patient blood was hybridized with probes containing hypervariable minicircle nucleotide sequences capable of detecting three sublineages of T. cruzi. Probes Z-I and Z-IIb detect unique sequences in lineages TcI and TcIIb, respectively. Probe Z-hybrid detects sequences of lineages TcIId and TcIIe. T. cruzi clones of the Z-I sublineage were detected in 62.2% of T. infestans dejections and 5.4% of peripheral blood samples. Clones of Z-IIb and Z-hybrid sublineages had similar distribution in blood and dejection samples. Interestingly, clones of the Z-IIb sublineage were significantly lower in cardiopathic than in non-cardiopathic patients (23.5% versus 75%; P=0.0006). Clones of the Z-hybrid sublineage were found in 29.4% of cardiopathic and 75% of non-cardiopathic patients, respectively (P=0.0051). By contrast, clones of sublineage Z-I were similarly distributed in both groups of patients. The low frequency of Z-IIb and Z-hybrid sublineage clones detected in cardiopathic patients suggests that the immunological mechanisms involved in controlling and eliminating these T. cruzi parasites may be detrimental to the host, leading to the development of chagasic cardiomyopathy.

Temporal variation of Trypanosoma cruzi infection in native mammals in Chile.

In the present study, we compared Trypanosoma cruzi infection in four native mammals from a hyperendemic area of Chagas disease in Chile for two different periods to assess the occurrence of interannual variation (1999-2000 vs. 2005-2006). Parasite detection in mammals is performed by polymerase chain reaction assays and confirmed by Southern blot analysis and hybridization test with a universal probe. Results showed significant differences in the levels of T. cruzi infection between the compared periods. We suggest that the major El Niño event occurred in 1997-1998, a large-scale global climatic fluctuation, could be indirectly explaining the extremely high T. cruzi infection in 1999-2000 by means of a time-lag response of the wild transmission cycle of Chagas disease in semiarid Chile after the irruption of small rodent populations.

Trypanosoma cruzi Genotypes in Mepraia gajardoi from Wild Ecotopes in Northern Chile

We evaluated Trypanosoma cruzi infection in 397 wild Mepraia gajardoi specimens from five coastal localities in northern Chile by detection of minicircle DNA by polymerase chain reaction. The wild capture sites were classified into two ecotopes: a fully wild ecotope (ecotope 1) and a wild ecotope near human dwellings (ecotope 2). Infection rates varied between 11% and 27%. Minicircle hybridization assays showed that T. cruzi lineages Tc II and Tc VI were commonly detected in ecotope 1 and ecotope 2, respectively. These results are discussed in the context of insect proximity to human dwellings, the alimentary profile of Mepraia sp., T. cruzi lineages detected in the past in the same disease-endemic area circulating in humans, and Triatoma infestans.

Wing shape differentiation of Mepraia species (Hemiptera: Reduviidae)

Mepraia is an endemic genus found in the semiarid and arid regions of north-central Chile. Until 1998, Mepraia spinolai was the only species of the genus, distributed in coastal and interior valleys from Chile between 18° and 34°S. However, on the basis of karyotype and morphological characters, coastal desert populations between 18° and 26°S were ranked as a new species, Mepraia gajardoi. Recently, genetic studies using nuclear and mitochondrial markers on Mepraia populations suggest that the geographical criterion to separate the two species should be reviewed. Mepraia species show conspicuous alary polymorphism, unique in the Triatominae subfamily. Females of both species are invariably micropterous, while males of M. spinolai can be micropterous, brachypterous or macropterous, and only brachypterous in M. gajardoi. In this study, we use geometric morphometrics analyses to compare male wings of M. spinolai and M. gajardoi from natural populations, in order to examine if these two species have diverged in alary shape. As expected, we found that brachypterous wings of both species are smaller than macropterous wings of M. spinolai. Additionally, we detected clear differences in shape on wings of M. gajardoi and M. spinolai, not attributable to allometric effects. For last, a new alary phenotype, insects with vestigial wings, was described here for the first time. In conclusion, our analyses on wings of Mepraia species separate two distinct groups consistent with the two described species. However, our findings of vestigial wings in some coastal areas of the north part of Chile cannot rule out the existence of a hybrid zone.