High phylogeographic structure in sylvatic vectors of Chagas disease of the genus Mepraia (Hemiptera: Reduviidae).

The hematophagous Hemiptera of the subfamily Triatominae are a very diverse group with a variety of morphs, behaviors and distributions. They have great epidemiological importance because many of its members are vectors of the protozoan Trypanosoma cruzi, the agent of Chagas disease. Mepraia is a genus of Triatominae endemic to Chile responsible for transmitting T. cruzi in the sylvatic cycle. Mepraia includes three species, M. gajardoi (18° 30'-26° 30' S) M. spinolai (26° 30'-34° 20' S) and the recently described M. parapatrica in intermediate zones (24° 36'-26° 51' S). Using mitochondrial DNA sequences, we inferred historical processes that led to the current structure of populations. Phylogeographic analyses identified three lineages, congruent with current taxonomy, and populations were highly structured. The times to the most recent common ancestor suggest that M. spinolai is the oldest lineage. We discuss the taxonomic and biogeographic implications of our results.

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.

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.

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.

Temporal fluctuation of infection with different Trypanosoma cruzi genotypes in the wild rodent Octodon degus.

We identified and followed-up for two years Octodon degus rodents infected with Trypanosoma cruzi genotypes by using xenodiagnosis with two vector species (Mepraia spinolai and Triatoma infestans), polymerase chain reaction DNA-based detection of insect dejections, Southern blot analysis, and minicircle hybridization with genotype-specific probes. Results show temporal fluctuations of infection with four parasite lineages (TCI, TCII, TCV, and TCVI) in one co-infected O. degus. Results are discussed in the context of parasitemia level and infection control in mammal hosts.

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.

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.

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.

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.

Coexistence of Trypanosoma cruzi genotypes in wild and periodomestic mammals in Chile.

Epidemiologic evidence suggests a preferential association of Trypanosoma cruzi genotypes TCI and TCII with marsupials and placental mammals, respectively. We identify T. cruzi genotypes from 117 infected mammals. Minicircle DNA amplified by polymerase chain reaction and hybridization with a panel of four specific probes showed frequencies for the T. cruzi genotypes TCI, TCIIb, TCIId, and TCIIe of 38%, 41%, 26%, and 9%, respectively, in wild mammals. In peridomestic mammals, frequencies for the same clones were 29%, 33%, 43%, and 14%, respectively. As a whole, mixed infections are found in more than 31% of the cases, which indicates the coexistence of multiclonal strains circulating in nature, and the absence of specific associations between T. cruzi genotypes and reservoir hosts, including marsupials. The direct characterization of parasite genotypes emphasizes the importance of obtaining unbiased epidemiologic information from parasite-endemic areas. Results are discussed in the context of competition or facilitation of T. cruzi genotypes within hosts.

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.

Comparison of Trypanosoma cruzi detection by PCR in blood and dejections of Triatoma infestans fed on patients with chronic Chagas disease.

In this study, we compare the sensitivity of detecting Trypanosoma cruzi in dejections of Triatoma infestans nymphs that had fed on the blood of chronic chagasic patients, with detection of T. cruzi in peripheral blood, using a polymerase chain reaction assay (PCR-D and PCR-B, respectively). Fifty-seven chronic patients were evaluated who were positive (group I) or negative (group II) by xenodiagnosis (XD). Patients showed 84.8 and 75% positive PCR results in both kinds of samples in groups I and II, respectively. Six cases (10.5%) showed positive PCR-D and negative PCR-B, five of them belonged to group I. In contrast, five cases of group II showed negative PCR-D and positive PCR-B. Overall, the PCR-D assay gave positive results in 52 out of 57 samples (91.2%), while 51 out of 57 (89.5%) were positive by PCR-B. In comparison, only 57.9% were positive by XD (p = 0.0001). In conclusion, PCR performed in dejection or blood was more sensitive for the parasite detection than xenodiagnosis. All patients (100%) were detected positive when both, PCR-D and PCR-B, were applied.

Variation in Trypanosoma cruzi clonal composition detected in blood patients and xenodiagnosis triatomines: implications in the molecular epidemiology of Chile.

To identify Trypanosoma cruzi clones from chronically infected individuals, they were transferred to triatomines by the xenodiagnosis test (XD) with Triatoma infestans. Polymerase chain reaction (PCR) and hybridization assays were performed to detect minicircle DNA in human blood samples and triatomine feces, using probes to determine the T. cruzi clones present. T. cruzi clone 19 (TcI) resulted the most prevalent in humans, with a frequency of 0.70 compared with a frequency of 0.53 in triatomines. T. cruzi clone 39 (TcIId) was the most prevalent in T. infestans, with a frequency of 0.65 compared with 0.33 in humans. The T. cruzi clone 43 (TcIIe) was not detected in blood samples; nevertheless, it was present at a rate of 0.17 in T. infestans feces. In conclusion, the T. cruzi clones are associated to each host, suggesting that selective amplification of clones occurs in human and triatomines.

Dissimilar distribution of Trypanosoma cruzi clones in humans after chemotherapy with allopurinol and itraconazole.

OBJECTIVES: The aim of this work was to study the distribution of Trypanosoma cruzi clones after treatment failure with itraconazole or allopurinol in infected humans. METHODS: Blood samples from treated and untreated individuals were used to detect T. cruzi by PCR assays and were confirmed by hybridization tests using total kinetoplast DNA as a universal probe. Also, xenodiagnosis (XD) tests were performed with Triatoma infestans fed from the same group of patients. We performed Southern-blot analyses of PCR products from blood or XD samples using a panel of four genotype-specific probes: corresponding to T. cruzi clones TcI, TcIIb, TcIId and TcIIe. The membranes were hybridized with radiolabelled probes and exposed in a Personal Molecular Imager. RESULTS: When comparing the presence of T. cruzi clones in the allopurinol-treated group with the non-treated group significant differences were only observed for XD samples. Clone TcI was present in 9/13 (69.2%) of the XD samples of the treated group, but only in 8/27 (29.6%) in the non-treated group (P = 0.0178). When the itraconazole-treated group and the control group were compared, significant differences were found in both the blood and XD samples. In blood, the clone TcIIb was detected in 6/17 (35.5%) of the treated group and in 18/27 (66.7%) of the non-treated group (P = 0.0207). When XD samples were analysed, the clone TcI was observed in 14/17 (82.3%) of the itraconazole-treated group but only in 8/27 (29.6%) of the control group (P = 0.0006), which suggests resistance of this clone to itraconazole. CONCLUSIONS: We detected a dissimilar distribution of T. cruzi clones in treated and untreated groups of patients. The presence of TcI increased in patients treated with allopurinol and itraconazole, whereas the presence of TcIIb decreased in itraconazole-treated patients. The type of T. cruzi clone that prevails suggests that TcI is resistant to both drugs and that TcIIb is susceptible to itraconazole.

Short report: Trypanosoma cruzi infection in wild mammals from a chagasic area of Chile.

We report results of PCR-DNA based detection of Trypanosoma cruzi in wild mammal reservoirs from a chagasic area of Chile. We analyzed 157 blood samples from wild mammals including the marsupial Thylamis elegans and the rodents Octodon degus, Phyllotis darwini, and Abrothrix olivaceus. In addition, 42 blood samples from goats (i.e., a peridomestic mammal) were analyzed. Blood samples were used to extract DNA, and PCR was performed using the amplification of minicircle DNA sequences. Southern analysis was used to confirm diagnosis with a universal probe of P(32)-labeled kinetoplast DNA. Altogether, 51% of the wild and 36% of the peridomestic mammals were infected with T. cruzi. These findings suggest that the real T. cruzi infection levels in wild and peridomiciliary reservoirs are higher than those previously determined with serological and parasitological conventional methods. The relevance of our results is discussed in term of the risk factor of infection in human.

Instability of Trypanosoma cruzi DNA in blood lysates: importance for PCR DNA-based diagnosis.

OBJECTIVE: In order to evaluate the stability of Trypanosoma cruzi kinetoplast DNA (kDNA), the blood samples from seven patients with Chagas disease were stored in different buffers and at different temperatures. METHODS: Three different buffers were used: buffer A, 6 mol/L guanidine-HCl; buffer B, 6M guanidine-HCl and 0.2M EDTA pH 7.5; and buffer C, 6M guanidine-HCl, 0.2M EDTA pH 7.5 and 10 microM dl-alpha-tocopherol (Roche, Basal, Switzerland). Two temperatures were used: 4 degrees C and 25 degrees C. Vitamin E was added to the blood lysates as an antioxidant. T. cruzi kDNA was obtained by phenol extraction, and then PCR amplifications and Southern blot were carried out in each DNA sample up to 90 days of blood storage. The iron content of each sample was determined by atomic absorption spectrophotometry. RESULTS: Overall, there is an association between T. cruzi kDNA stability and the storage time of blood samples. No significant differences were detected in T. cruzi kDNA stability in the presence or absence of vitamin E or with citrate or EDTA as an anticoagulant. There was no statistical difference in the failure of PCR-based kDNA detection with these different storage buffers, temperatures or iron levels. CONCLUSIONS: The blood lysates promote T. cruzi kDNA damage in a time-dependent manner that reduces the ability to detect the genomic DNA of an infectious agent by PCR. The high concentration of guanidine-HCl denatured proteins in these storage conditions probably denotes a non-enzymatic kDNA lysis.