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New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis
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.
Journal Article
First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease
Real-Time PCR (qPCR) testing is recommended as both a diagnostic and outcome measurement of etiological treatment in clinical practice and clinical trials of Chagas disease (CD), but no external quality assurance (EQA) program provides performance assessment of the assays in use. We implemented an EQA system to evaluate the performance of molecular biology laboratories involved in qPCR based follow-up in clinical trials of CD. An EQA program was devised for three clinical trials of CD: the E1224 (NCT01489228), a pro-drug of ravuconazole; the Sampling Study (NCT01678599), that used benznidazole, both conducted in Bolivia; and the CHAGASAZOL (NCT01162967), that tested posaconazole, conducted in Spain. Four proficiency testing panels containing negative controls and seronegative blood samples spiked with 1, 10 and 100 parasite equivalents (par. eq.)/mL of four Trypanosoma cruzi stocks, were sent from the Core Lab in Argentina to the participating laboratories located in Bolivia and Spain. Panels were analyzed simultaneously, blinded to sample allocation, at 4-month intervals. In addition, 302 random blood samples from both trials carried out in Bolivia were sent to Core Lab for retesting analysis. The analysis of proficiency testing panels gave 100% of accordance (within laboratory agreement) and concordance (between laboratory agreement) for all T. cruzi stocks at 100 par. eq./mL; whereas their values ranged from 71 to 100% and from 62 to 100% at 1 and 10 par. eq./mL, respectively, depending on the T. cruzi stock. The results obtained after twelve months of preparation confirmed the stability of blood samples in guanidine-EDTA buffer. No significant differences were found between qPCR results from Bolivian laboratory and Core Lab for retested clinical samples. This EQA program for qPCR analysis of CD patient samples may significantly contribute to ensuring the quality of laboratory data generated in clinical trials and molecular diagnostics laboratories of CD.
Journal Article
Dietary indexes based on the EAT-Lancet recommendations: a narrative review
Background The publication of the EAT-Lancet Commission in 2019 has motivated the development of numerous dietary indexes to evaluate how different populations adhere to its proposed dietary guidelines aimed at promoting both human and planetary health. However, the lack of methodological harmonization among these indexes limits their comparability and applicability in population-level assessments. This narrative review aims to synthesize the existing EAT-Lancet-based indexes, identify limitations, and propose criteria to develop a new index. Methods We conducted a non-systematic narrative search to identify dietary indexes based on the EAT-Lancet recommendations and published up to April 24, 2025. The information was synthesized in a comparative table, including scoring structure, the food groups considered, and the identified limitations. Results We identified thirteen original indexes and three systematic reviews evaluating eleven of these indexes. Analysis of the indexes show important common limitations: the use of the same score for each category, not considering the EAT-Lancet recommended ranges, grouping of food items with very different environmental impacts (such as beef and pork), penalizing vegetarian diets, and not penalizing unhealthy foods such as alcohol, cocoa, and coffee. A new index is proposed which weights every food group according to four environmental dimensions, gives maximum scores within the recommended ranges, separates cheese from the rest of the dairy, allows for interchangeability between protein and fat sources and it penalizes unhealthy foods and total caloric deviations. Conclusions This review confirms the methodological heterogeneity across thirteen dietary indexes based on the EAT-Lancet recommendations. We propose the creation of a new standardized index to address the limitations of previous indexes and foster greater consistency in future studies, and better applicability of planetary principles across diverse populations. Key messages • A standardized method to measure the dietary environmental impact is needed to support effective strategies that benefit both public and planetary health. • This review highlights major methodological differences among the existing EAT-Lancet-based indexes and the need for harmonization to ensure comparability across diverse populations.
Journal Article
Autoimmune encephalopathy in Graves’ disease: remission after total thyroidectomy
According to the recently proposed criteria of Tamagno and colleagues, 3 a diagnosis of SREAT was made and the patient was treated with intravenous methylprednisolone 1 g once a day for 5 days, with excellent improvement of his clinical conditions: only minor distal tremors were observed and the EEG was normal. [...]we would like to conclude suggesting that serum antithyroid antibodies, independently of their pathogenic role, may be considered as potentially useful markers for monitoring SREAT progression; their levels may also be considered in those acute or subacute encephalopathies where a clear pathogenesis is missing, even in the presence of an euthyroid state.
Journal Article