BackgroundA century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.Methodology/FindingsAn international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.Conclusion/SignificanceThis study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Fifteen cases of human pseudoterranovosis are reported for Chile, representing an emerging parasitic infection in this country caused by larvae of the nematode Pseudoterranova sp. Our observations also included an outbreak of pseudoterranovosis in 3 of 4 individuals who shared the same raw fish dish (cebiche). Most of the cases occurred in adult patients. The main source of infection was from consumption raw or fried marine fish, including hakes (Merluccius australis or Merlucciuts gayi), pomfret (Brama australis), Inca scad (Trachurus murphvi), and corvina (Cilus gilberti). Seasonal distribution showed most of the cases to occur in fall and spring. Parasite larvae were isolated from the mouths of most of the patients after they reported a pharyngeal tickling sensation, coughing, vomiting, or a foreign body in the mouth or throat.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite’s genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite.

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.

To investigate whether Trypanosoma cruzi populations found in chagasic cardiopathic and non-cardiopathic patients are genetically differentiated, three molecular microsatellite markers were analysed. This analysis was also applied to compare T. cruzi samples from peripheral blood or dejections of Triatoma infestans fed on the blood of the same patients. In order to obtain the first objective, analyses of predominant T. cruzi genotypes were conducted using three approaches: a locus-by-locus analysis; a Fisher method across three loci; and analysis of molecular variance by Genepop and Arlequin programs. Only with one locus and on the blood samples was a significant differentiation detected among non-cardiopathic and cardiopathic groups, which was not confirmed by the other two methods. On the contrary, with the three approaches, it was found that T. cruzi clones present in the blood of patients are genetically differentiated from those detected in dejections of T. infestans fed on the same patients. Our results showed that the most frequent lineage both in blood as well as in triatomine dejection samples was TcI. No significant difference in T. cruzi lineage distribution was observed among chagasic cardiopathic and non-cardiopathic patients. The majority of the samples (50-60%) had only one T. cruzi clone (uniclonal) either in blood or dejection samples.