TcTASV Antigens of Trypanosoma cruzi: Utility for Diagnosis and High Accuracy as Biomarkers of Treatment Efficacy in Pediatric Patients.

The discovery and characterization of novel parasite antigens to improve the diagnosis of Trypanosoma cruzi by serological methods and for accurate and rapid follow-up of treatment efficiency are still needed. TcTASV is a T. cruzi-specific multigene family, whose products are expressed on the parasite stages present in the vertebrate host. In a previous work, a mix of antigens from subfamilies TcTASV-A and TcTASV-C (Mix A + C) was sensitive and specific to identify dogs with active infection of high epidemiological relevance. Here, TcTASV-A and TcTASV-C were assayed separately as well as together (Mix A + C) in an ELISA format on human samples. The Mix A + C presented moderate sensitivity (78%) but high diagnostic accuracy with a 100% of specificity, evaluated on healthy, leishmaniasic, and Strongyloides stercoralis infected patients. Moreover, antibody levels of pediatric patients showed-2 years posttreatment-diminished reactivity against the Mix A + C (P < 0.0001), pointing TcTASV antigens as promising tools for treatment follow-up.

Epidemiology of American Tegumentary Leishmaniasis and Trypanosoma cruzi Infection in the Northwestern Argentina.

Background. Endemic areas of tegumentary leishmaniasis (TL) in Salta, Argentina, present some overlap zones with the geographical distribution of Chagas disease, with mixed infection cases being often detected. Objectives. The purpose of this study was to determine the magnitude of Leishmania sp. infection and potential associated risk factors, the serologic prevalence of T. cruzi, and the presence of T. cruzi-Leishmania sp. mixed infection in a region of the northwest of Argentina. Methods. Cross-sectional studies were conducted to detect TL prevalence and T. cruzi seroprevalence. A case-control study was conducted to examine leishmaniasis risk factors. Results. Prevalence of TL was 0.17%, seroprevalence of T. cruzi infection was 9.73%, and mixed infection proportion-within the leishmaniasic patients group-was 16.67%. The risk factors associated with TL transmission were sex, age, exposure to bites at work, staying outdoors more than 10 hours/day, bathing in the river, and living with people who had lesions or were infected during the study. Discussion. The endemic pattern of TL seems to involve exposure of patients to vectors in wild as well as peridomestic environment. Cases of T. cruzi infection are apparently due to migration. Therefore, a careful epidemiological surveillance is necessary due to the contraindication of antimonial administration to chagasic patients.

Experimental evidence of biological interactions among different isolates of Trypanosoma cruzi from the Chaco Region.

Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region.

Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.

Preponderant clonal evolution of Trypanosoma cruzi I from Argentinean Chaco revealed by Multilocus Sequence Typing (MLST).

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI.

Optimized multilocus sequence typing (MLST) scheme for Trypanosoma cruzi.

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.

Response of Solanum tuberosum to Myzus persicae infestation at different stages of foliage maturity.

Young leaves of the potato Solanum tuberosum L. cultivar Kardal contain resistance factors to the green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) and normal probing behavior is impeded. However, M. persicae can survive and reproduce on mature and senescent leaves of the cv. Kardal plant without problems. We compared the settling of M. persicae on young and old leaves and analyzed the impact of aphids settling on the plant in terms of gene expression. Settling, as measured by aphid numbers staying on young or old leaves, showed that after 21 h significantly fewer aphids were found on the young leaves. At earlier time points there were no difference between young and old leaves, suggesting that the young leaf resistance factors are not located at the surface level but deeper in the tissue. Gene expression was measured in plants at 96 h postinfestation, which is at a late stage in the interaction and in compatible interactions this is long enough for host plant acceptance to occur. In old leaves of cv. Kardal (compatible interaction), M. persicae infestation elicited a higher number of differentially regulated genes than in young leaves. The plant response to aphid infestation included a larger number of genes induced than repressed, and the proportion of induced versus repressed genes was larger in young than in old leaves. Several genes changing expression seem to be involved in changing the metabolic state of the leaf from source to sink.

Comparative analysis of Solanum stoloniferum responses to probing by the green peach aphid Myzus persicae and the potato aphid Macrosiphum euphorbiae.

Plants protect themselves against aphid attacks by species-specific defense mechanisms. Previously, we have shown that Solanum stoloniferum Schlechtd has resistance factors to Myzus persicae Sulzer (Homoptera: Aphididae) at the epidermal/mesophyll level that are not effective against Macrosiphum euphorbiae Thomas (Homoptera: Aphididae). Here, we compare the nymphal mortality, the pre-reproductive development time, and the probing behavior of M. persicae and M. euphorbiae on S. stoloniferum and Solanum tuberosum L. Furthermore, we analyze the changes in gene expression in S. stoloniferum 96 hours post infestation by either aphid species. Although the M. euphorbiae probing behavior shows that aphids encounter more probing constrains on phloem activities-longer probing and salivation time- on S. stoloniferum than on S. tuberosum, the aphids succeeded in reaching a sustained ingestion of phloem sap on both plants. Probing by M. persicae on S. stoloniferum plants resulted in limited feeding only. Survival of M. euphorbiae and M. persicae was affected on young leaves, but not on senescent leaves of S. stoloniferum. Infestation by M. euphorbiae changed the expression of more genes than M. persicae did. At the systemic level both aphids elicited a weak response. Infestation of S. stoloniferum plants with a large number of M. persicae induced morphological changes in the leaves, leading to the development of pustules that were caused by disrupted vascular parenchyma and surrounding tissue. In contrast, an infestation by M. euphorbiae had no morphological effects. Both plant species can be regarded as good host for M. euphorbiae, whereas only S. tuberosum is a good host for M. persicae and S. stoloniferum is not. Infestation of S. stoloniferum by M. persicae or M. euphorbiae changed the expression of a set of plant genes specific for each of the aphids as well as a set of common genes.

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina.

The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance.

Interest and limitations of Spliced Leader Intergenic Region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco.

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference.