Trypanosoma Cruzi Discrete Typing Units Research Articles

First report of mixed Trypanosoma cruzi discrete typing units infection in Triatoma phyllosoma in the peri-urban environment of Oaxaca, Mexico.

Chagas disease, a zoonosis transmitted mainly by hematophagous insects of the subfamily Triatominae, is caused by Trypanosoma cruzi, classified into six discrete typing units (DTUs: TcI-TcVI and Tcbat). Insect vectors were collected from 84 human dwellings in the municipality of Santo Domingo Tehuantepec, Oaxaca, Mexico; 4.76% were infested. DTUs were determined using conventional and nested PCR. The infection rate was 43.6%. All insects were infected with TcI while one specimen showed mixed infection with TcII. This is the first report of T. cruzi mixed infection in Triatoma phyllosoma, its main vector in the study region.

High Parasitic Loads Quantified in Sylvatic Triatoma melanica, a Chagas Disease Vector.

Triatoma melanica is a sylvatic vector species in Brazil. In We aimed to characterize the Trypanosoma cruzi discrete typing units (DTUs), the parasitic loads, and the blood meal sources of insects collected in rocky outcrops in rural areas in the state of Minas Gerais. An optical microscope (OM) and kDNA-PCR were used to examine natural infection by T. cruzi, and positive samples were genotyped by conventional multilocus PCR. Quantification of the T. cruzi load was performed using qPCR, and the blood meal sources were identified by Sanger sequencing the 12S rRNA gene. A total of 141 T. melanica were captured. Of these, ~55% (61/111) and ~91% (63/69) were positive by OM and KDNA-PCR, respectively. We genotyped ~89% (56/63) of the T. cruzi-positive triatomines, with TcI (~55%, 31/56) being the most prevalent DTU, followed by TcIII (~20%, 11/56) and TcII (~7%, 4/56). Only TcI+TcIII mixed infections were detected in 10 (~18%) specimens. A wide range of variation in the parasitic loads of T. melanica was observed, with an overall median value of 104 parasites/intestine, with females having higher T. cruzi loads than N2, N4, and N5. TcII showed lower parasitic loads compared to TcI and TcIII. The OM positive diagnosis odds ratio between T. cruzi infection when the parasite load is 107 compared to 103 was approximately 29.1. The most frequent blood meal source was Kerodon rupestris (~58%), followed by Thrichomys apereoides (~18%), Wiedomys cerradensis (~8%), Galactis cuja (~8%) and Gallus gallus (~8%). Our findings characterize biological and epidemiological aspects of the sylvatic population of T. melanica in the study area, highlighting the need to extend surveillance and control to this vector.

Congenital Chagas disease in Santa Cruz Department, Bolivia, is dominated by Trypanosoma cruzi lineage V.

This study identified Trypanosoma cruzi discrete typing units (DTUs) in maternal and infant specimens collected from two hospitals in Bolivia, using conventional genotyping and DTU-specific serotyping. Specimens from 142 mothers were used, including 24 seronegative and 118 seropositive individuals; 29 women transmitted T. cruzi to their infants. Maternal and infant parasite loads were determined by quantitative real-time PCR. Maternal sera were tested with an in-house parasite lysate ELISA and serotyped by a lineage-specific peptide ELISA, targeting the trypomastigote small surface antigen (TSSA). Trypanosoma cruzi genotypes in infected infants were determined by a triple PCR-RFLP assay. All infant specimens were genotyped as TcV. Maternal parasite loads and absorbance values by the lysate ELISA were significantly higher for transmitters compared with non-transmitters. Among seropositive mothers, 65.3% had positive results by the TSSA II/V/VI peptide ELISA. No significant difference in reactivity to TSSA II/V/VI was observed for transmitters compared with non-transmitters (79.3% vs 60.7%, respectively). Our findings reinforce the difficulty in obtaining sufficient sample numbers and parasite DNA to investigate the interaction between parasite genetics and the risk of congenital transmission and argue for the inclusion of DTU-specific serotyping in prospective studies.

Shelter cats host infections with multiple Trypanosoma cruzi discrete typing units in southern Louisiana

Trypanosoma cruzi is a zoonotic parasite endemic in the southern US and the Americas, which may frequently infect dogs, but limited information is available about infections in cats. We surveyed a convenience sample of 284 shelter cats from Southern Louisiana to evaluate T. cruzi infection using serological and PCR tests. Parasites from PCR positive cats were also genotyped by PCR and deep sequencing to assess their genetic diversity. We detected a seropositivity rate for T. cruzi of at least 7.3% (17/234), and 24.6% of cats (70/284) were PCR positive for the parasite. Seropositivity increased with cat age (R2 = 0.91, P = 0.011), corresponding to an incidence of 7.2% ± 1.3 per year, while PCR positivity decreased with age (R2 = 0.93, P = 0.007). Cats were predominantly infected with parasites from TcI and TcVI DTUs, and to a lesser extent from TcIV and TcV DTUs, in agreement with the circulation of these parasite DTUs in local transmission cycles. These results indicate that veterinarians should have a greater awareness of T. cruzi infection in pets and that it would be important to better evaluate the risk for spillover infections in humans.

Association between Trypanosoma cruzi DTU TcII and chronic Chagas disease clinical presentation and outcome in an urban cohort in Brazil.

The specific roles of parasite characteristics and immunological factors of the host in Chagas disease progression and prognosis are still under debate. Trypanosoma cruzi genotype may be an important determinant of the clinical chronic Chagas disease form and prognosis. This study aimed to identify the potential association between T. cruzi genotypes and the clinical presentations of chronic Chagas disease. This is a retrospective study using T. cruzi isolated from blood culture samples of 43 patients with chronic Chagas disease. From 43 patients, 42 were born in Brazil, mainly in Southeast and Northeast Brazilian regions, and one patient was born in Bolivia. Their mean age at the time of blood collection was 52.4±13.2 years. The clinical presentation was as follows 51.1% cardiac form, 25.6% indeterminate form, and 23.3% cardiodigestive form. Discrete typing unit (DTU) was determined by multilocus conventional PCR. TcII (n = 40) and TcVI (n = 2) were the DTUs identified. DTU was unidentifiable in one patient. The average follow-up time after blood culture was 5.7±4.4 years. A total of 14 patients (32.5%) died and one patient underwent heart transplantation. The cause of death was sudden cardiac arrest in six patients, heart failure in five patients, not related to Chagas disease in one patient, and ignored in two patients. A total of 8 patients (18.6%) progressed, all of them within the cardiac or cardiodigestive forms. TcII was the main T. cruzi DTU identified in chronic Chagas disease Brazilian patients (92.9%) with either cardiac, indeterminate or cardiodigestive forms, born at Southeast and Northeast regions. Other DTU found in much less frequency was TcVI (4.8%). TcII was also associated to patients that evolved with heart failure or sudden cardiac arrest, the two most common and ominous consequences of the cardiac form of Chagas disease.

Mixed infections by different Trypanosoma cruzi discrete typing units among Chagas disease patients in an endemic community in Panama.

BackgroundTrypanosoma cruzi, the hemoparasite that causes Chagas disease, is divided into six Discrete Typing Units or DTUs: TcI-TcVI plus Tcbat. This genetic diversity is based on ecobiological and clinical characteristics associated with particular populations of the parasite. The main objective of this study was the identification of DTUs in patients with chronic chagasic infections from a mountainous rural community in the eastern region of Panama.MethodsA total of 106 patients were tested for Chagas disease with three serological tests (ELISA, rapid test, and Western blot). Molecular diagnosis and DTU typing were carried out by conventional PCRs and qPCR targeting different genomic markers, respectively. As a control sample for the typing, 28 patients suspected to be chagasic from the metropolitan area of Panama City were included.ResultsResults showed a positivity in the evaluated patients of 42.3% (33/78); high compared to other endemic regions in the country. In the control group, 20/28 (71.43%) patients presented positive serology. The typing of samples from rural patients showed that 78.78% (26/33) corresponded to TcI, while 9.09% (3/33) were mixed infections (TcI plus TcII/V/VI). Seventy-five percent (15/20) of the patients in the control group presented TcI, and in five samples it was not possible to typify the T. cruzi genotype involved.ConclusionsThese results confirm that TcI is the main DTU of T. cruzi present in chronic chagasic patients from Panama. However, the circulation of other genotypes (TcII/V/VI) in this country is described for the first time. The eco-epidemiological characteristics that condition the circulation of TcII/V/VI, as well as the immune and clinical impact of mixed infections in this remote mountainous region should be investigated, which will help local action programs in the surveillance, prevention, and management of Chagas disease.

Exploring the parasite load and molecular diversity of Trypanosoma cruzi in patients with chronic Chagas disease from different regions of Brazil.

Chagas disease is still a major public health issue in many Latin American countries. One of the current major challenges is to find an association between Trypanosoma cruzi discrete typing units (DTUs) and clinical manifestations of the disease. In this study, we used a multilocus conventional PCR and quantitative real time PCR (qPCR) approaches to perform the molecular typing and parasite load quantification directly from blood specimens of 65 chronic Chagas disease patients. All patients were recruited at the same health center, but their place of birth were widely distributed in different geographic regions of Brazil. Of the 65 patients, 35 (53.8%) presented positive amplification by real time qPCR, being 20 (30.7%) with the clinical indeterminate form and 15 (23.1%) with the cardiac form of the disease. The parasite load median for all positive patients was 2.54 [1.43–11.14] parasite equivalents/mL (par. Eq./mL), with the load ranging from 0.12 to 153.66 par. Eq./mL. Noteworthy, the parasite load was significantly higher in patients over 70 years old (median 20.05 [18.29–86.86] par. Eq./mL). Using guanidine-EDTA blood samples spiked with reference T. cruzi strains, belonging to the six DTUs, it was possible to genotype the parasite up to 0.5 par. Eq./mL, with high specificity. Of the patients with positive qPCR, it was possible to identify the T. cruzi DTU in 28 patients (80%). For the remaining patients (20%), at least a partial result was obtained. Analysis of specimens showed prevalences of TcVI, TcII and mixed infection TcVI+TcII equal to 40%, 17.1% and 14.3%, respectively. In addition, two patients were infected by TcV, and one patient was coinfected by TcIII+TcVI, These last three patients were in stage A of chronic chagasic cardiomyopathy (CCC), and they were born at the Bahia State (northeast region of Brazil). When T. cruzi genotypes were compared with the parasite load, more elevated parasite loads were observed in patients infected by TcII in general (parasite load median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL). However, while the frequency of CCC was 50% in patients infected by TcVI and TcV, only 16.7% of patients infected by TcII evolved to CCC. Taking together, our results contribute to update the epidemiological knowledge of T. cruzi DTUs in Brazil, and highlight the age of patient and infection by TcII as important features that lead to the observation of higher parasitemia levels.

Comparing vector competence of Mepraia gajardoi and Triatoma infestans by genotyping Trypanosoma cruzi discrete typing units present in naturally infected Octodon degus

Chagas disease is a vector-borne disease caused by the parasite Trypanosoma cruzi, and transmitted by triatomine insects to several mammal species. In Chile, the wild triatomine species are the endemic Mepraia species, and the only domestic vector of Chagas disease is Triatoma infestans. The aim of this study was to determine the competence of M. gajardoi compared to T. infestans as a T. cruzi vector using the naturally infected rodent Octodon degus. M. gajardoi amplified T. cruzi present in all O. degus studied while T. infestans only in half of the infected rodents. Both triatomine species excrete metacyclic trypomastigotes and amplified the same three T. cruzi DTUs, however, M. gajardoi showed differences in their ability to amplify TcI. TcV and TcVI had the same probability to be amplified by both triatomine species. Both species amplified mixed infections, with TcI-TcVI as the most represented. This study reports the higher vector competence of M. gajardoi in comparison to T. infestans.

Molecular characterization of Trypanosoma cruzi samples derived from Triatoma vitticeps and Panstrongylus geniculatus of the Atlantic rainforest, southeast Brazil

Background: In rural areas of Espírito Santo state, southeast Brazil, triatomine species attracted by light frequently invade residences. The aim of this study was to investigate the Trypanosoma cruzi discrete typing units (DTUs) harbored by these triatomines. Methods: Triatomine’s intestinal contents were examined, inoculated in mice, and the positive samples were cultivated. Flagellates obtained from infected mice hemoculture were submitted to DNA extraction using a salting-out method and to TcSC5D gene amplification. The amplified samples were sequenced, and polymorphism was analyzed for DTU identification. Results: Three hundred and ninety-four triatomines were identified: Triatoma vitticeps (90.03%), Panstrongylus geniculatus (8.89%), Panstrongylus megistus (0.54%), Panstrongylus diasi (0.27%), and Triatoma tibiamaculata (0.27%). Among the specimens, 251/394 (67.65%) presented flagellated forms similar to T. cruzi. After triatomine intestinal content inoculation into mice, 134 mice presented T. cruzi-like trypomastigotes from Tr. vitticeps and P. geniculatus and 89 samples were positive in hemoculture. Sixty-two samples were analyzed for the TcSC5D gene and TcI, TcII, TcIII, and TcIV DTUs were identified. Conclusions: We observed T. cruzi DTU diversity in Tr. vitticeps and P. geniculatus, which showed the predominance of TcII and occurrence of TcI, TcIII and TcIV. Triatomines presented high T. cruzi infection rates. Since little is known regarding the possible mammalian hosts that maintain the T. cruzi cycle, further studies are necessary to obtain a better understanding of the parasite transmission cycle in this region.

Analysis of over 1500 triatomine vectors from across the US, predominantly Texas, for Trypanosoma cruzi infection and discrete typing units

Across the Americas, triatomine insects harbor diverse strains of Trypanosoma cruzi (T. cruzi), agent of Chagas disease. Geographic patterns of vector infection and parasite strain associations, especially in vectors encountered by the public, may be useful in assessing entomological risk, but are largely unknown across the US. We collected Triatoma spp. from across the US (mainly Texas), in part using a citizen science initiative, and amplified T. cruzi DNA to determine infection prevalence and parasite discrete typing units (DTUs). We found 54.4% infection prevalence in 1510 triatomines of 6 species; prevalence in adult T. gerstaeckeri (63.3%; n=897) and T. lecticularia (66.7%; n=66) was greater than in T. sanguisuga (47.6%; n=315), T. indictiva (47.8% n=67), T. rubida (14.1%; n=64), and T. protracta (10.5%; n=19). The odds of infection in adults were 9.73 times higher than in nymphs (95% CI 4.46–25.83). PCR of the spliced leader intergenic region (SL-IR) and/or the putative lathosterol/episterol oxidase TcSC5D gene revealed exclusively T. cruzi DTUs TcI and TcIV; 5.5% of T. cruzi-positive samples were not successfully typed. T. gerstaeckeri (n=548) were more frequently infected with TcI (53.9%) than TcIV (34.4%), and 11.9% showed mixed TcI/TcIV infections. In contrast, T. sanguisuga (n=135) were more frequently infected with TcIV (79.3%) than TcI (15.6%), and 5.2% showed mixed infections. Relative abundance of parasite DTUs varied spatially, with both TcI and TcIV co-circulating in vectors in central Texas, while TcIV predominated in northern Texas. Given prior findings implicating TcI in human disease and TcI and TcIV in animal disease in the US, knowledge of spatial distribution of T. cruzi infection and DTUs in vectors is important to understanding public and veterinary health risk of T. cruzi infection.

Cytochrome c oxidase subunit 1 gene as a DNA barcode for discriminating Trypanosoma cruzi DTUs and closely related species

BackgroundThe DNA barcoding system using the cytochrome c oxidase subunit 1 mitochondrial gene (cox1 or COI) is highly efficient for discriminating vertebrate and invertebrate species. In the present study, we examined the suitability of cox1 as a marker for Trypanosoma cruzi identification from other closely related species. Additionally, we combined the sequences of cox1 and the nuclear gene glucose-6-phosphate isomerase (GPI) to evaluate the occurrence of mitochondrial introgression and the presence of hybrid genotypes.MethodsSixty-two isolates of Trypanosoma spp. obtained from five of the six Brazilian biomes (Amazon Forest, Atlantic Forest, Caatinga, Cerrado and Pantanal) were sequenced for cox1 and GPI gene fragments. Phylogenetic trees were reconstructed using neighbor-joining, maximum likelihood, parsimony and Bayesian inference methods. Molecular species delimitation was evaluated through pairwise intraspecific and interspecific distances, Automatic Barcode Gap Discovery, single-rate Poisson Tree Processes and multi-rate Poisson Tree Processes.ResultsBoth cox1 and GPI genes recognized and differentiated T. cruzi, Trypanosoma cruzi marinkellei, Trypanosoma dionisii and Trypanosoma rangeli. Cox1 discriminated Tcbat, TcI, TcII, TcIII and TcIV. Additionally, TcV and TcVI were identified as a single group. Cox1 also demonstrated diversity in the discrete typing units (DTUs) TcI, TcII and TcIII and in T. c. marinkellei and T. rangeli. Cox1 and GPI demonstrated TcI and TcII as the most genetically distant branches, and the position of the other T. cruzi DTUs differed according to the molecular marker. The tree reconstructed with concatenated cox1 and GPI sequences confirmed the separation of the subgenus Trypanosoma (Schizotrypanum) sp. and the T. cruzi DTUs TcI, TcII, TcIII and TcIV. The evaluation of single nucleotide polymorphisms (SNPs) was informative for DTU differentiation using both genes. In the cox1 analysis, one SNP differentiated heterozygous hybrids from TcIV sequences. In the GPI analysis one SNP discriminated Tcbat from TcI, while another SNP distinguished TcI from TcIII.ConclusionsDNA barcoding using the cox1 gene is a reliable tool to distinguish T. cruzi from T. c. marinkellei, T. dionisii and T. rangeli and identify the main T. cruzi genotypes.

Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) in Latin-American migrants in Barcelona (Spain)

Trypanosoma cruzi, the causative agent of Chagas disease, is divided into six Discrete Typing Units (DTUs): TcI–TcVI. We aimed to identify T. cruzi DTUs in Latin-American migrants in the Barcelona area (Spain) and to assess different molecular typing approaches for the characterization of T. cruzi genotypes. Seventy-five peripheral blood samples were analyzed by two real-time PCR methods (qPCR) based on satellite DNA (SatDNA) and kinetoplastid DNA (kDNA). The 20 samples testing positive in both methods, all belonging to Bolivian individuals, were submitted to DTU characterization using two PCR-based flowcharts: multiplex qPCR using TaqMan probes (MTq-PCR), and conventional PCR. These samples were also studied by sequencing the SatDNA and classified as type I (TcI/III), type II (TcII/IV) and type I/II hybrid (TcV/VI). Ten out of the 20 samples gave positive results in the flowcharts: TcV (5 samples), TcII/V/VI (3) and mixed infections by TcV plus TcII (1) and TcV plus TcII/VI (1). By SatDNA sequencing, we classified the 20 samples, 19 as type I/II and one as type I. The most frequent DTU identified by both flowcharts, and suggested by SatDNA sequencing in the remaining samples with low parasitic loads, TcV, is common in Bolivia and predominant in peripheral blood. The mixed infection by TcV–TcII was detected for the first time simultaneously in Bolivian migrants. PCR-based flowcharts are very useful to characterize DTUs during acute infection. SatDNA sequence analysis cannot discriminate T. cruzi populations at the level of a single DTU but it enabled us to increase the number of characterized cases in chronically infected patients.

Trypanosoma cruzi diversity in infected dogs from areas of the north coast of Chile

As part of a multi-site research program on the eco-epidemiology and control of Chagas disease in northern Chile, we sought to identify the Trypanosoma cruzi discrete typing units (DTUs) infecting rural and peridomestic dogs, using direct methods without grown of the parasite in the laboratory and thus to assess the use of this species as a sentinel of the disease in well-defined endemic areas of T. cruzi in Chile. Infected dogs (35) from three villages were included in the study. The studied villages were Caleta Río Seco and Caleta San Marcos, both in the Tarapacá Region, and La Serena in the Coquimbo Region. These villages were selected based on previous evidence of Mepraia infection reports of the Chilean Ministry of Health. Amplicons from nested-PCR positive samples were used as targets to determine the infective T. cruzi DTUs circulating in blood using PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). Results of hybridization with dog samples from Caleta Rio Seco showed single infections in 2 out of 16 and mixed infections in 14 out of 16. TcVI was the most frequent DTU found in this area. A highlight is that for the first time the presence of TcIII is reported in this area. Samples from Caleta San Marcos showed single infections in 5 out of 9 and mixed infections in 4 out of 9. TcVI was the most frequent DTU found in this area. Samples from La Serena showed single infections in 5 out of 10 and mixed infections in 2 out of 10; we were unable to genotype the other 3 samples. Our results indicate that infection by T. cruzi DTUs in dogs is not homogeneously distributed but rather specific to each region of our country, as demonstrated by the differences in the T. cruzi DTU distribution in some localities.

Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples

Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa=0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed.

Fluctuations in Trypanosoma cruzi discrete typing unit composition in two naturally infected triatomines: Mepraia gajardoi and M. spinolai after laboratory feeding

Mepraia species are hematophagous insects and the most important wild vectors of Trypanosoma cruzi, the causative agent of Chagas disease in southeastern South America. Because the domestic Triatoma infestans is already controlled, the transmission of different T. cruzi discrete typing units (DTUs) by Mepraia species deserves attention. Our aim is to gather information on the diversity of T. cruzi DTUs circulating in natural insect populations.Two groups of naturally infected bugs 21 Mepraia gajardoi and 26 Mepraia spinolai were followed-up after two or more laboratory feedings by means of minicircle-PCR assays to evaluate the composition of four T. cruzi DTUs by hybridization tests. Fluctuations from positive T. cruzi detection to negative and the converse, as well as single to mixed infections with different T. cruzi DTUs and the opposite were frequent observations after laboratory feeding in both Mepraia species. Single and mixed infections with more than two T. cruzi DTUs were detected after the first feeding; however mainly mixed infections prevailed after the second feeding. Laboratory feeding on three or more occasions resulted in a decreasing trend of the parasite burden. In a comparison with 28 infected and fed M. gajardoi collected one year before from the same vector colony T. cruzi DTUs composition changed, indicating that temporal variations occur in T. cruzi.Natural populations of Mepraia species can transmit complex mixtures T. cruzi DTUs which fluctuate over time after feeding, with a tendency to eliminate the parasitism after prolonged feeding.

Genotyping of Trypanosoma cruzi DTUs and Trypanosoma rangeli genetic groups in experimentally infected Rhodnius prolixus by PCR-RFLP

The specific detection and genetic typing of trypanosomes that infect humans, mammalian reservoirs, and vectors is crucial for diagnosis and epidemiology. We utilized a PCR-RFLP assay that targeted subunit II of cytochrome oxidase and 24Sα-rDNA to simultaneously detect and discriminate six Trypanosoma cruzi discrete typing units (DTUs) and two genetic groups of Trypanosoma rangeli (KP1+/KP1−) in intestinal contents of experimentally infected Rhodnius prolixus. The PCR assays showed that in 23 of 29 (79.4%) mixed infections with the six T. cruzi DTUs and mixed infections with individual DTUs and/or groups KP1+ and KP1−, both parasites were successfully detected. In six mixed infections that involved TcIII, the TcI, TcII, TcV, and TcVI DTUs predominated to the detriment of TcIII, indicating the selection of genetic groups. Interactions between different genetic groups and vectors may lead to genetic selection over TcIII. The elimination of this DTU by the immune system of the vector appears unlikely because TcIII was present in other mixed infections (TcIII/TcIV and TcIII/KP1+). Both molecular markers used in this study were sensitive and specific, demonstrating their usefulness in a wide geographical area where distinct genotypes of these two species are sympatric. Although the cellular and molecular mechanisms that are involved in parasite-vector interactions are still poorly understood, our results indicate a dynamic selection toward specific T. cruzi DTUs in R. prolixus during mixed genotype infections.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Direct molecular identification of Trypanosoma cruzi Discrete Typing Units in domestic and peridomestic Triatoma infestans and Triatoma sordida from the Argentine Chaco

We assessed the distribution of Trypanosoma cruzi Discrete Typing Units (DTUs) in domestic and peridomestic Triatoma infestans and Triatoma sordida specimens collected in a well-defined rural area in Pampa del Indio, northeastern Argentina. Microscopically-positive bugs were randomly selected with a multi-level sampling design, and DTUs were identified using direct PCR strategies. TcVI predominated in 61% of 69 T. infestans and in 56% of 9 T. sordida. TcV was the secondary DTU in T. infestans (16%) and was found in 1 T. sordida specimen (11%). Three T. sordida (33%) were found infected with TcI, a DTU also identified in local Didelphis albiventris opossums. Mixed DTU infections occurred rarely (5%) and were detected both directly from the bugs' rectal ampoule and parasite cultures. The identified DTUs and bug collection sites of T. infestans were significantly associated. Bugs infected with TcV were almost exclusively captured in domiciles whereas those with TcVI were found similarly in domiciles and peridomiciles. All mixed infections occurred in domiciles. TcV-infected bugs fed more often on humans than on dogs, whereas TcVI-infected bugs showed the reverse pattern. T. sordida is a probable sylvatic vector of TcI linked to D. albiventris, and could represent a secondary vector of TcVI and TcV in the domestic/peridomestic cycle.

Genetic subdivisions within Trypanosoma cruzi (Discrete Typing Units) and their relevance for molecular epidemiology and experimental evolution

BackgroundThis paper summarizes the main results obtained on Trypanosoma cruzi genetic diversity and population structure since this parasite became the theme of many genetic and molecular studies in the early seventies.ResultsT. cruzi exibits a paradigmatic pattern of long-term, clonal evolution, which has structured its natural populations into several discrete genetic subdivisions or "Discrete Typing Units" (DTU). Rare hybridization events are nevertheless detectable in natural populations and have been recently obtained in the laboratory.ConclusionsThe DTUs and natural clones of T. cruzi constitute relevant units for molecular epidemiology and experimental evolution. Experimental mating opens the way to an in-depth knowledge of this parasite's formal genetics.