Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity

El análisis del polimorfismo genético es una poderosa herramienta para la vigilancia epidemiológica y investigar. Sin embargo, la inferencia poderosa de la variación genética del patógeno es a menudo restringido por el acceso limitado al ADN objetivo representativo, especialmente en el estudio de especies parásitas obligadas para las cuales el cultivo ex vivo requiere muchos recursos o es propenso a sesgos. Los métodos modernos de captura de secuencias permiten analizar directamente la variación genética de los patógenos del material del huésped/vector, pero a menudo son demasiado complejos y costosos para entornos de escasos recursos donde prevalecen las enfermedades infecciosas. Este estudio propone un método sencillo y rentable Herramienta de tipificación de secuencias de locus de todo el genoma (GLST) basada en la amplificación paralela masiva de puntos críticos de información en todo el genoma del patógeno objetivo. el multiplexado La reacción en cadena de la polimerasa amplifica cientos de objetivos genéticos diferentes definidos por el usuario en un único tubo de reacción y la posterior limpieza basada en gel de agarosa y código de barras completan la preparación de la biblioteca por menos de 4 USD por muestra. Nuestro estudio genera un modelo flexible Flujo de trabajo de diseño de panel de imprimación GLST para Trypanosoma cruzi, el agente parásito de Chagas enfermedad. Aplicamos con éxito nuestro panel GLST de 203 objetivos a extractos nómicos metagénicos directos y sin cultivo de vectores triatominos que contienen un mínimo de 3,69 pg/μl de ADN de T. cruzi y elaborar más sobre el rendimiento del método mediante la secuenciación de bibliotecas GLST de T. cruzi clones de referencia que representan unidades de tipificación discretas (DTU) TcI, TcIII, TcIV, TcV y TcVI. Los 780 sitios SNP que identificamos en el conjunto de muestras distinguen parásitos de forma repetitiva infectar vectores simpátricos y detectar correlaciones entre distancias genéticas y geográficas a escala regional (< 150 km), así como continental. Los marcadores también separan claramente TcI, TcIII, TcIV y TcV + TcVI y parecen distinguir infecciones multiclonales dentro de TcI. Discutimos las ventajas, limitaciones y perspectivas de nuestro método a través de un espectro de la investigación epidemiológica.Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obli gate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Mod ern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor set tings where infectious diseases prevail. This study proposes a simple, cost-effective ‘genome-wide locus sequence typing’ (GLST) tool based on massive parallel amplifica tion of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding com pletes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metage nomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic dis tances at regional (< 150 km) as well as continental scales. The markers also clearly sep arate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research

Psammolestes arthuri naturalmente infectado con trypanosoma cruzi encontrado en simpatría con rhodnius prolixus y triatoma maculata en nidos de aves en el estado Anzoátegui, Venezuela | Psammolestes arthuri naturally infected with trypanosoma cruzi found in sympatry with rhodnius prolixus and triatoma maculata on bird nests in Anzoátegui state, Venezuela

RESUMENEn Venezuela, la enfermedad de Chagas es un problema de salud pública con aproximadamente dos millones de personas infectadas y más de seis millones en riesgo de contraer la patología. En esta investigación se comprobó la presencia del chinche triatómido, Psammolestes arthuri, en nidos de diferentes especies de aves en comunidades rurales del estado Anzoátegui, algunos de ellos infectados naturalmente con Trypanosoma cruzi, en simpatria con otros triatominos (Rhodnius prolixus y Triatoma maculata). Se recolectaron 3.277 triatominos en 478 nidos de seis especies de aves (Phacellodomus rufifrons, Troglodytes aedon, Icterus icterus, I. nigrogularis, Cacicus cela y Psarocolius decumanus). Se encontró que 99,05% (3.246/3.277) eran ejemplares de Ps. arthuri y 0,95% (31/3.277) de otras especies de triatominos, dentro de las cuales 0,57% (19/3.277) era R. prolixus y 0,37%(12/3.277) T. maculata. De los Ps. arthuri encontrados, 0,12% (4/3.246) presentaron infección natural por T. cruzi. La caracterización parasitológica de uno de los aislados de T. cruzy en ratones albinos machos de la cepa MNRI, mostró alta afinidad por células de musculatura cardiaca, esquelética y lisa, con una parasitemia máxima de 2,4 x 104 parásitos/mL de formas sanguícolas de T. cruzi y una mortalidad de 100% de los ratones inoculados. Este aislado fue tipificado molecularmente como perteneciente al genotipo TcIII. Los resultados muestran que en el estado Anzoátegui, P. arthuri es una especie que se alimenta predominantemente de sangre de aves con bajo riesgo de transmisión vectorial de la enfermedad de Chagas a humanos. Palabras clave: Hemiptera, Reduviidae, Triatominae, Kinetoplastida, Trypanosomatidae, Aves,Passeriformes. ABSTRACTIn Venezuela, Chagas' disease is a public health problem with around 2 million people infected and more than 6 million under risk of infection. In this study the presence of the triatomid Psammolestes arthuri is reported in nests of different species of birds from rural communities of Anzoátegui State, some of them found naturally infected with Trypanosoma cruzi, in sympatry with other species of triatomines (Rhodnius prolixus y Triatoma maculata). A total of 3,277 triatomine specimens were collected in 478 nests from 6 species of birds (Phacellodomus rufifrons, Troglodytes aedon, Icterus icterus, I. nigrogularis, Cacicus cela y Psarocolius decumanus). It was found that 99.05% (3246/3277) of specimens were P. arthuri and 0.95% (31/3277) other triatomine species, fromwhich 0.57% (19/3277) were R. prolixus and 0.37% (12/3277) T. maculata. Only 0.12% (4/3246) of P. arthuri were infected with T. cruzi. The parasitological characterization of one T. cruzi isolate in white male NMRI mice showed high affinity for cardiac, skeletal and smooth muscle cells, with a peak parasitemia of 2.4 x 104 parasites/ mL blood stream forms of T. cruzi and 100% mortality of inoculated mice. This isolate was molecularly typed as belonging to TcIII genotype. The results show that in Anzoátegui State, P. arthuri predominantly feed on blood of birds, representing a low risk for vector transmission of Chagas' disease to humans. Key words: Hemiptera, Reduviidae, Triatominae, Kinetoplastida, Trypanosomatidae, Aves, Passeriformes

Geographical Distribution of Trypanosoma cruzi Genotypes in Venezuela

Chagas disease is an endemic zoonosis native to the Americas and is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The parasite is also highly genetically diverse, with six discrete typing units (DTUs) reported TcI – TcVI. These DTUs broadly correlate with several epidemiogical, ecological and pathological features of Chagas disease. In this manuscript we report the most comprehensive evaluation to date of the genetic diversity of T. cruzi in Venezuela. The dataset includes 778 samples collected and genotyped over the last twelve years from multiple hosts and vectors, including nine wild and domestic mammalian host species, and seven species of triatomine bug, as well as from human sources. Most isolates (732) can be assigned to the TcI clade (94.1%); 24 to the TcIV group (3.1%) and 22 to TcIII (2.8%). Importantly, among the 95 isolates genotyped from human disease cases, 79% belonged to TcI - a DTU common in the Americas, however, 21% belonged to TcIV- a little known genotype previously thought to be rare in humans. Furthermore, were able to assign multiple oral Chagas diseases cases to TcI in the area around the capital, Caracas. We discuss our findings in the context of T. cruzi DTU distributions elsewhere in the Americas, and evaluate the impact they have on the future of Chagas disease control in Venezuela

Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research

The miniJPAS survey: clusters and galaxy groups detection with AMICO

Samples of galaxy clusters allow us to better understand the physics at play in galaxy formation and to constrain cosmological models once their mass, position (for clustering studies) and redshift are known. In this context, large optical data sets play a crucial role. We investigate the capabilities of the Javalambre-Physics of the Accelerating Universe Astrophysical Survey (J-PAS) in detecting and characterizing galaxy groups and clusters. We analyze the data of the miniJPAS survey, obtained with the JPAS-Pathfinder camera and covering $1$ deg$^2$ centered on the AEGIS field to the same depths and with the same 54 narrow band plus 2 broader band near-UV and near-IR filters anticipated for the full J-PAS survey. We use the Adaptive Matched Identifier of Clustered Objects (AMICO) to detect and characterize groups and clusters of galaxies down to $S/N=2.5$ in the redshift range $0.05<z<0.8$. We detect 80, 30 and 11 systems with signal-to-noise ratio larger than 2.5, 3.0 and 3.5, respectively, down to $\sim 10^{13}\,M_{\odot}/h$. We derive mass-proxy scaling relations based on Chandra and XMM-Newton X-ray data for the signal amplitude returned by AMICO, the intrinsic richness and a new proxy that incorporates the galaxies' stellar masses. The latter proxy is made possible thanks to the J-PAS filters and shows a smaller scatter with respect to the richness. We fully characterize the sample and use AMICO to derive a probabilistic membership association of galaxies to the detected groups that we test against spectroscopy. We further show how the narrow band filters of J-PAS provide a gain of up to 100% in signal-to-noise ratio in detection and an uncertainty on the redshift of clusters of only $\sigma_z=0.0037(1+z)$ placing J-PAS in between broadband photometric and spectroscopic surveys. The performances of AMICO and J-PAS with respect to mass sensitivity, mass-proxies qualityComment: 15 pages, 12 figures, 3 tables, submitted to A&

J-PLUS: Toward a homogeneous photometric calibration using Gaia BP/RP low-resolution spectra

Aims. We present the photometric calibration of the 12 optical passbands for the Javalambre Photometric Local Universe Survey (J-PLUS) third data release (DR3) comprising 1642 pointings of two square degrees each. Methods. We selected nearly 1.5 million main sequence stars with a signal-to-noise ratio larger than ten in the 12 J-PLUS passbands and available low-resolution (R = 20–80) spectrum from the blue and red photometers (BP/RP) in Gaia DR3. We compared the synthetic photometry from BP/RP spectra with the J-PLUS instrumental magnitudes after correcting for the magnitude and color terms between both systems in order to obtain a homogeneous photometric solution for J-PLUS. To circumvent the current limitations in the absolute calibration of the BP/RP spectra, the absolute color scale was derived using the locus of 109 white dwarfs closer than 100 pc with a negligible interstellar extinction. Finally, the absolute flux scale was anchored to the Panoramic Survey Telescope and Rapid Response System (Pan-STARRS) photometry in the r band. Results. The precision of the J-PLUS photometric calibration estimated from duplicated objects observed in adjacent pointings and by comparison with the spectro-photometric standard star GD 153 is ~12 mmag in u, J0378, and J0395, and it is ~7 mmag in J0410, J0430, ɡ, J0515, r, J0660, i, J0861, and z. The estimated accuracy in the calibration along the surveyed area is better than 1% for all the passbands. Conclusions. The Gaia BP/RP spectra provide a high-quality, homogeneous photometric reference in the optical range across the full sky in spite of their current limitations as an absolute reference. The calibration method for J-PLUS DR3 reaches an absolute precision and accuracy of 1% in the 12 optical filters within an area of 3284 square degrees

Repeat-Driven Generation of Antigenic Diversity in a Major Human Pathogen, Trypanosoma cruzi.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure

Research priorities for neglected infectious diseases in Latin America and the Caribbean Region

Global priorities for research in neglected infectious diseases (NIDs) can be assessed in different ways, but it is important to realize that regional priorities may significantly differ one from another. The region of Latin America and the Caribbean (LAC) is—along with Africa and Asia—more affected by NIDs than other regions of the world. Some of the Latin American NIDs are common to other continents, while others are very specific or disproportionately affect the Latin American region [1– 3] (Table 1). Because of its huge ecological diversity, ongoing environmental changes, and massive migrations, LAC is also a catalyst for the (re-)emergence and spreading of NIDs, both inside and outside the subcontinent. Following a colloquium on NIDs in LAC held in Lima, Peru, between 12 and 14 November 2009, a thematic workshop was organized with the support of the European Commission (EC). It involved 29 scientists (16 from the Americas, two from the Democratic Republic of Congo and India, respectively, and nine from Europe) working on different NIDs and representing several research areas from basic to applied. This report summarizes the consensus comments of the expert group after oral and written consultation. It is envisaged that this document should stimulate a debate within the scientific community and serve as a recommendation for future actions by international or regional funding agencies in the area of NIDs in LAC. (Párrafo extraído del texto a modo de resumen)Facultad de Ciencias Médica

Genotyping of Trypanosoma cruzi: systematic selection of assays allowing rapid and accurate discrimination of all known lineages.

Trypanosoma cruzi, the agent of Chagas disease, can be subdivided into six discrete typing units (DTUs), TcI, TcIIa, TcIIb, TcIIc, TcIId or TcIIe, each having distinct epidemiologically important features. Dozens of genetic markers are available to determine the DTU to which a T. cruzi isolate belongs, but there is no consensus on which should be used. We selected five assays: three polymerase chain reaction (PCR)-restriction fragment length polymorphisms based on single nucleotide polymorphisms (SNPs) in the HSP60, Histone H1, and GPI loci, and PCR product size polymorphism of the LSU rDNA and mini-exon loci. Each assay was tested for its capacity to differentiate between DTUs using a panel of 48 genetically diverse T. cruzi clones. Some markers allowed unequivocal identification of individual DTUs, however, only by using a combination of multiple markers could all six DTUs be resolved. Based upon the results we recommend a triple-assay comprising the LSU rDNA, HSP60 and GPI markers for reliable, rapid, low-cost DTU assignment