Course of serological tests in treated subjects with chronic Trypanosoma cruzi infection: A systematic review and meta-analysis of individual participant data.

OBJECTIVE: To determine the course of serological tests in subjects with chronic Trypanosoma cruzi infection treated with anti-trypanosomal drugs. METHODS: A systematic review and meta-analysis was conducted using individual participant data. Survival analysis and the Cox proportional hazards regression model with random effects to adjust for covariates were applied. The protocol was registered in the PROSPERO database (http://www.crd.york.ac.uk/PROSPERO; CRD42012002162). RESULTS: A total of 27 studies (1296 subjects) conducted in eight countries were included. The risk of bias was low for all domains in 17 studies (63.0%). Nine hundred and thirteen subjects were assessed (149 seroreversion events, 83.7% censored data) for enzyme-linked immunosorbent assay (ELISA), 670 subjects (134 events, 80.0% censored) for indirect immunofluorescence assay (IIF), and 548 subjects (99 events, 82.0% censored) for indirect hemagglutination assay (IHA). A higher probability of seroreversion was observed within a shorter time span in subjects aged 1-19 years compared to adults. The chance of seroreversion also varied according to the country where the infection might have been acquired. For instance, the pooled adjusted hazard ratio between children/adolescents and adults for the IIF test was 1.54 (95% confidence interval 0.64-3.71) for certain countries of South America (Argentina, Bolivia, Chile, and Paraguay) and 9.37 (95% confidence interval 3.44-25.50) for Brazil. CONCLUSIONS: The disappearance of anti-T. cruzi antibodies was demonstrated along the course of follow-up. An interaction between age at treatment and country setting was found.

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.

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

BACKGROUND: A 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/FINDINGS: An 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/SIGNIFICANCE: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

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.