In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

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A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

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The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications.

Summary: Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and causes potentially life-threatening disease of the heart and gastrointestinal tract. The southern half of the United States contains enzootic cycles of T. cruzi, involving 11 recognized triatomine vector species. The greatest vector diversity and density occur in the western United States, where woodrats are the most common reservoir; other rodents, raccoons, skunks, and coyotes are also infected with T. cruzi. In the eastern United States, the prevalence of T. cruzi is highest in raccoons, opossums, armadillos, and skunks. A total of 7 autochthonous vector-borne human infections have been reported in Texas, California, Tennessee, and Louisiana; many others are thought to go unrecognized. Nevertheless, most T. cruzi-infected individuals in the United States are immigrants from areas of endemicity in Latin America. Seven transfusion-associated and 6 organ donor-derived T. cruzi infections have been documented in the United States and Canada. As improved control of vector- and blood-borne T. cruzi transmission decreases the burden in countries where the disease is historically endemic and imported Chagas' disease is increasingly recognized outside Latin America, the United States can play an important role in addressing the altered epidemiology of Chagas' disease in the 21st century.

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Trypanosoma cruzi and Chagas' Disease in the United States

American trypanosomiasis (Chagas disease).

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.

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

Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia

Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.

Clonal propagation is considered to be the predominant mode of reproduction among many parasitic protozoa. However, this assumption may overlook unorthodox, infrequent or cryptic sexuality. Trypanosoma cruzi, which causes Chagas disease, is known to undergo non-Mendelian genetic exchange in the laboratory. In the field, evidence of extant genetic exchange is limited. In this study, we undertook intensive sampling of T. cruzi Discrete Typing Unit I in endemic eastern Colombia. Using Fluorescence-activated cell sorting, we generated 269 biological clones from 67 strains. Each clone was genotyped across 24 microsatellite loci. Subsequently, 100 representative clones were typed using 10 mitochondrial sequence targets (3.76 Kbp total). Clonal diversity among humans, reservoir hosts and vectors suggested complex patterns of superinfection and/or coinfection in oral and vector-borne Chagas disease cases. Clonal diversity between mother and foetus in a congenital case demonstrates that domestic TcI genotypes are infective in utero. Importantly, gross incongruence between nuclear and mitochondrial markers is strong evidence for widespread genetic exchange throughout the data set. Furthermore, a confirmed mosaic maxicircle sequence suggests intermolecular recombination between individuals as a further mechanism of genetic reassortment. Finally, robust dating based on mitochondrial DNA indicates that the emergence of a widespread domestic TcI clade that we now name TcI(DOM) (formerly TcIa/VEN(Dom)) occurred 23 000 ± 12 000 years ago and was followed by population expansion, broadly corresponding with the earliest human migration into the Americas.

/pdf/contemporary-cryptic-sexuality-in-trypanosoma-cruzi-55jd0r2od7.pdf

Contemporary cryptic sexuality in Trypanosoma cruzi

Background 
Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes.


Methodology and Principal Findings 
High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission.


Conclusions 
These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed.

/pdf/molecular-epidemiology-of-human-oral-chagas-disease-ccfhme8gzo.pdf

Molecular epidemiology of human oral Chagas disease outbreaks in Colombia.

SummaryChagas disease is an endemic disease of the American continent caused by Try-panosoma cruzi and divided into six discrete typing units (TcI – TcVI). Nearly10 million people harbour the infection representing a serious issue in publichealth. Epidemiological surveillance allowed us to detect a bat-related T. cruzigenotype (henceforth named TcBat) in a 5-year-old female living in a forest areain northwestern Colombia. Molecular tools determined a mixed infection ofT. cruzi I and TcBat genotypes. This represents the ﬁrst report of TcBat infectionin humans; the epidemiological consequences of this ﬁnding are discussed herein.Chagas disease or American trypanosomiasis, caused byTrypanosoma cruzi, is a complex neglected tropical diseasethat affects over 8 million people in Latin America (Sch-munis and Yadon, 2010). This parasite can be transmittedvia contaminated faeces of triatomine bugs, oral route,blood transfusion, congenital route, laboratory accidentsand transplantation (Rassi et al., 2012). The aetiologicalagent is characterized by exhibiting a remarkable geneticvariability evinced in at least six discrete typing units(DTUs) from T. cruzi ItoT. cruzi VI (TcI-TcVI) (Zingaleset al., 2012). Recently, intensive sampling has reported anew genotype associated with bats from the Noctilio spp.,Myotis spp. and Artibeus spp. species named as TcBat (Mar-cili et al., 2009; Pinto et al., 2012). The role of TcBat inhuman infections is unclear; previous studies have demon-strated its ability to infect murine models and invadeHe-La culture lines, but there have not yet to be any reportsof this genotype associated with human cases (Maeda et al.,2011).In 2012, a screening for acute Chagas disease was carriedout in Colombia as part of the National Health Institutesurveillance system. Twenty-six patients from the munici-pality of Sabana del Crespo of the department of Cesar innortheastern Colombia were evaluated. This communitylives in the middle of the dry forest, in rudimentary houseswhere they interact with the fauna of the region (triatominebugs, mammals, etc.). We conducted serological tests onthe 26 patients using crude antigenic preparations ofT. cruzi I by ELISA, IFAT and HAI commercial kit (Wie-ner, Argentina), obtaining a positivity of 19% with a fatalcase of death due to acute cardiomyopathy. When entomo-logical and epidemiological surveillance was conducted, weobserved the presence of adults of Rhodnius prolixus andTriatoma dimidiata inside the houses and adults ofTriatoma maculata and Panstrongylus geniculatus aroundthe dwellings. Previous entomological surveillance in themunicipality has reported an infestation index of 50%.Likewise, the presence of mammals (rodents, didelphidsand dogs) within the houses and the dwellings wasobserved.Blood samples were taken to seed haemocultures andPCR directed to kDNA using primers previously reported

Marleny Montilla

Papers

Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia

Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.

Contemporary cryptic sexuality in Trypanosoma cruzi

Molecular epidemiology of human oral Chagas disease outbreaks in Colombia.

First report of human Trypanosoma cruzi infection attributed to TcBat genotype.