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

Bontempi, Iván; Bizai, María Laura; Ortíz, Sylvia; Manattini, Silvia; Fabbro, Diana Lucrecia; Solari, Aldo; Diez, Cristina

doi:10.1016/j.meegid.2016.05.026

Cited by 13 publications

(12 citation statements)

References 62 publications

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“…Although T. cruzi DTUs have been successfully identified from whole blood, the probability of successful identification decreases markedly in chronic patients and with increasing age (Bontempi et al, 2016;Burgos et al, 2007;Cura et al, 2012;Lucero et al, 2016). All isolates here reported were from chronic patients, and 25% belonged to people aged ≥ 18 years.…”

Section: Accepted Manuscriptmentioning

confidence: 83%

“…In Argentina, the most frequent DTU identified in humans is TcV, followed by TcVI. Notably, other DTU prevalent among T. cruzi-infected patients in other Latin American countries such as TcI and TcII have rarely been found in Argentina (Bontempi et al, 2016; Bua Burgos et al, 2010;Cardinal et al, 2008;Cura et al, 2012;Diez et al, 2010;Diosque et al, 2003;Lucero et al, 2016;Monje-Rumi et al, 2013. Mixed infections (i.e., more than one DTU identified in a single patient) have been less frequently reported than in Bolivia and Chile (R. Del Puerto et al, 2010;Solari et al, 2001), and were mainly associated with Chagas disease reactivation due to immunosuppression (Burgos et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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New human isolates of Trypanosoma cruzi confirm the predominance of hybrid lineages in domestic transmission cycle of the Argentinean Chaco

Macchiaverna

Enriquez

Buscaglia

et al. 2018

Infection, Genetics and Evolution

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Trypanosoma cruzi, the etiological agent of Chagas disease, was initially classified into 6 Discrete Typing Units (DTUs). The hybrid DTUs TcV and TcVI are the most frequent in domestic transmission cycles throughout the Southern Cone countries of South America. Here, we genotyped parasite isolates from human residents in Pampa del Indio municipality, Chaco, to further characterize the structure of T. cruzi populations, and to assess the degree of overlapping between the domestic and sylvatic transmission cycles. Artificial xenodiagnostic tests were performed to blood samples from 125 T. cruzi-seropositive people (age range, 3-70 years) who represented 14.3% of all seropositive residents identified. Parasites were obtained from feces of T. cruzi-infected Triatoma infestans examined 30 or 60 days after blood-feeding, and grown in vitro. The cultured parasites were genotyped by means of two PCR-based protocols. DTUs were determined from 39 (31%) patients residing in 28 dwellings. The only DTUs identified were TcV (92%) and TcVI (8-36%). Households with more than one parasite isolate consistently displayed the same DTU. Further sequencing of a fragment of the TcMK gene from selected samples argue against the occurrence of mixed TcV-TcVI infections in the study population. Sequencing data revealed an unexpected degree of genetic variability within TcV including two apparently robust subgroups of isolates. Our results for human residents confirm the predominance of hybrid lineages (TcV and to a much lesser extent TcVI) and the absence of sylvatic genotypes (TcI and TcIII) in (peri)domestic transmission cycles in the Argentinean Chaco area.

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Section: Accepted Manuscriptmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

New human isolates of Trypanosoma cruzi confirm the predominance of hybrid lineages in domestic transmission cycle of the Argentinean Chaco

Macchiaverna

Enriquez

Buscaglia

et al. 2018

Infection, Genetics and Evolution

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“…Based on the polymorphism of minicircle hypervariable regions, a highly repetitive sequence from kinetoplastid DNA (kDNA), a minicircle lineage-specific PCR assay has been developed to detect the presence of single or mixed infections of TcI, TcII, TcV and TcVI in clinical samples [ 50 , 51 ]. Considering that the analysis of SatDNA or kDNA sequences does not allow the identification of all T .…”

Section: Discussionmentioning

confidence: 99%

New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis

et al. 2017

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Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs), TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA), comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.

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“…This highlights that assessment of the ability of lead compounds to display in vivo activity against a wide panel of isolates, reflecting the diverse phylogeny and geographical range of T. cruzi , must be considered an integral step in the drug development pathway. The importance of this is emphasized further by the commonality of mixed infections (Bontempi et al 2016 ).…”

Section: Does Parasite Diversity Impact On Drug Efficacy?mentioning

confidence: 99%

Biological factors that impinge on Chagas disease drug development

et al. 2017

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SUMMARYChagas disease is caused by infection with the insect-transmitted protozoan Trypanosoma cruzi, and is the most important parasitic infection in Latin America. The current drugs, benznidazole and nifurtimox, are characterized by limited efficacy and toxic side-effects, and treatment failures are frequently observed. The urgent need for new therapeutic approaches is being met by a combined effort from the academic and commercial sectors, together with major input from not-for-profit drug development consortia. With the disappointing outcomes of recent clinical trials against chronic Chagas disease, it has become clear that an incomplete understanding of parasite biology and disease pathogenesis is impacting negatively on the development of more effective drugs. In addition, technical issues, including difficulties in establishing parasitological cure in both human patients and animal models, have greatly complicated the assessment of drug efficacy. Here, we outline the major questions that need to be addressed and discuss technical innovations that can be exploited to accelerate the drug development pipeline.

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

Cited by 13 publications

References 62 publications

New human isolates of Trypanosoma cruzi confirm the predominance of hybrid lineages in domestic transmission cycle of the Argentinean Chaco

New human isolates of Trypanosoma cruzi confirm the predominance of hybrid lineages in domestic transmission cycle of the Argentinean Chaco

New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis

Biological factors that impinge on Chagas disease drug development

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