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Native Americans from the Amazon, Andes, and coastal geographic regions of South America have a rich cultural heritage but are genetically understudied, therefore leading to gaps in our knowledge of their genomic architecture and demographic history. In this study, we sequence 150 genomes to high coverage combined with an additional 130 genotype array samples from Native American and mestizo populations in Peru. The majority of our samples possess greater than 90% Native American ancestry, which makes this the most extensive Native American sequencing project to date. Demographic modeling reveals that the peopling of Peru began -~12,000 y ago, consistent with the hypothesis of the rapid peopling of the Americas and Peruvian archeological data. We find that the Native American populations possess distinct ancestral divisions, whereas the mestizo groups were admixtures of multiple Native American communities that occurred before and during the Inca Empire and Spanish rule. In addition, the mestizo communities also show Spanish introgression largely following Peruvian Independence, nearly 300 y after Spain conquered Peru. Further, we estimate migration events between Peruvian populations from all three geographic regions with the majority of between-region migration moving from the high Andes to the low-altitude Amazon and coast. As such, we present a detailed model of the evolutionary dynamics which impacted the genomes of modern-day Peruvians and a Native American ancestry dataset that will serve as a beneficial resource to addressing the underrepresentation of Native American ancestry in sequencing studies. [ABSTRACT FROM AUTHOR]

Latent tuberculosis infection (LTBI) is defined as an infection with Mycobacterium tuberculosis (MTB) without clinical, bacteriological, or radiological findings, and its early diagnosis is essential for eradication of tuberculosis. To identify LTBI, we measured the numbers of interferon-? producing T cells, based on the ELISPOT assay, and the antibody titers in the sera to tuberculous glycolipid antigen (TBGL-Ab). Seventeen culture-confirmed TB patients, 13 controls from TB endemic areas (EC) and 13 controls from TB non-endemic areas (NEC) were enrolled. Peripheral blood mononuclear cells (2.5 x 105 per well) were cultured on plates precoated with antibody against interferon-γ. ELISPOT response was defined as positive when the MTB-specific antigen-containing wells showed at least 6 spots and twice numbers of spots than negative control wells. ELISPOT responses were positive in 15 (88%), 8 (62%) and 4 (31%) subjects of TB, EC and NEC groups, respectively. The ELISPOT data differ between TB and NEC groups (p < 0.01) but not between TB and EC groups. In contrast, TBGL-Ab titers were elevated (> 2.0 U/ml) in 12 TB patients (71%), but only in one subject (8%) each from EC and NEC groups. These results indicate the high prevalence of LTBI in EC. In conclusion, LTBI is associated with positive ELISPOT assay and the low titer of TBGL-Ab, while positive results both in ELISPOT and TBGL-Ab assays indicate active TB. The low titer of TBGL-Ab is a helpful marker to identify LTBI in ELISPOT-positive individuals in TB endemic areas. [ABSTRACT FROM AUTHOR]

Objetivo: analizar curvas de melting (High Resolution Melting: HRM) para diagnóstico de TB-MDR en muestras de esputo. Métodos: estudio retrospectivo, 250 muestras de esputo descontaminados fueron colectadas: 124 sensibles a rifampicina (RIF) e isoniacida (INH), 24 monoresistentes a RIF, 33 monoresistentes a INH y 69 multidrogo-resistentes. Extrajimos el ADN con kit comercial (Invitrogen). El PCR en tiempo real se realizó utilizando kit comercial (Master Mix 1X HRM, Qiagen) bajo las condiciones de ciclaje del fabricante. Cada muestra se corrió por duplicado para los genes rpoB, katG e inhA. Se usó ADN de la cepa H37Rv como referencia en los análisis. El análisis de HRM se realizó en el software del termociclador Rotogene Q (Qiagen). Se evaluaron los parámetros de sensibilidad y especificidad del análisis HRM en comparación con el método de referencia agar proporciones en placa (APP). Resultados: los análisis de HRM comparados con el APP mostraron una sensibilidad de 89,4 % y una especificidad de 90,4 % para la detección de TB-MDR en muestras de esputos descontaminados, considerando a los genes rpoB, katG y la región promotora inhA. Estos valores son mayores cuando se analizan los parámetros de sensibilidad y especificidad por separado para cada gen. Los valores de sensibilidad y especificidad estuvieron dentro del rango del intervalo de confianza de 95%. Las muestras analizadas por duplicado dieron el mismo resultado para cada uno de los marcadores evaluados: genes rpoB, katG e inhA, indicando un 100% de reproducibilidad. Conclusiones: el análisis de curvas de melting (HRM) mostró una adecuada sensibilidad y especificidad en el diagnóstico de TB-MDR. Su uso podría ser una buena alternativa al diagnóstico microbiológico, con esto se reduciría el tiempo de exposición de pacientes a personas sanas y el oportuno inicio de tratamiento de la enfermedad. [ABSTRACT FROM AUTHOR]

identificar la expresión de microARNs en pacientes diagnosticados con APCNP, tuberculosis (TB) activa y TB latente en comparación con controles sanos. Métodos: El trabajo siguió el diseño de un estudio mixto: prospectivo (pacientes con APCNP), retrospectivo (pacientes con diagnóstico TB latente y TB activa) y descriptivo. Un total de 14 muestras de suero fueron seleccionadas, de los cuales cuatro fueron controles sanos, tres pacientes con diagnóstico de TB latente, tres pacientes con diagnóstico de TB activa y cuatro pacientes con diagnóstico de APCNP. Se utilizó la metodología RNAseq para el secuenciamiento de ARN total a partir de suero e investigar la expresión diferencial de microARNs. Las librerías fueron analizadas en el secuenciador de próxima generación Hi-seq (Illumina Inc.). El análisis de datos a partir de las corridas de RNA-seq se realizó con programas bioinformáticos que nos permitieron identificar la expresión diferencial de microARNs en cada uno de los grupos analizados. Resultados: Se identificaron marcadas diferencias en la expresión de genes de ARNs presentes en controles sanos, pacientes con TB latente, TB activa y APCNP. Considerando un p-valor < 0.01 se identificaron un total de 2708 genes expresados diferencialmente. En APCNP (1873 genes), TB activa (159 genes) y TB latente (141 genes) fueron expresados diferencialmente en comparación con los controles sanos. Cuando se comparó el APCNP con TB activa y TB latente, se identificaron 275 y 259 genes expresados respectivamente. Así mismo, se identifica 1 solo gen expresado diferencialmente entre TB activa y TB latente. En el análisis de expresión diferencial de microARNs exosomales, se identificaron 3 microARNs específicos para APCNP: miR-200A, miR-485 y miR-7977 que fueron los más sobre-expresados en comparación con los controles sanos. Conclusiones: miR-200, miR-485 y miR- 7977 podrían ser usados como potenciales biomarcadores para el diagnóstico temprano del APCNP en países con alta incidencia de TB como el nuestro. Consideramos necesario realizar estudios adicionales para validar la posible aplicación de estos microARNs exosomales en respuesta al tratamiento y monitoreo de la enfermedad. [ABSTRACT FROM AUTHOR]

Lung cancer is a leading cause of cancer-related death among both men and women in the United States, where non-small cell lung cancer accounts for ~85% of lung cancer. Lung adenocarcinoma (ADC) is the major histologic subtype. The presence of actionable mutations prompts the use of therapies designed to specifically address the deleterious effects of those cancer-driving mutations; these therapies have already shown promise in cases carrying those actionable mutations (~30%). Innovative therapeutic approaches are needed for the treatment of 70% of patients suffering from lung ADC. Adoptive transfer of CD8+ T cells specific against cancer/testis (CT) Ags, whose protein expression is restricted to the gonads (testis and ovary) and cancerous cells, is an excellent alternative. In this study, we report the isolation of HLA-A*02:01/CT37 peptide-specific α and β TCR chains from a CD8+ T cell clone obtained from a patient suffering from lung ADC. We also report the development of an innovative CD3ζ construct. With those TCR chains and the engineered (modified) CD3ζ chain, we produced a construct that when transduced into CD8+ T cells is capable of redirecting transduced CD8+ T cell cytotoxic activity and IFN-γ secretion against peptide-pulsed autologous cells and HLA-A*02:01-positive and CT37-expressing lung ADC cell lines. Our findings will launch the development of innovative adoptive transfer immunotherapies for the treatment of lung ADC, targeting the most prevalent HLA molecules and CT37 peptides restricted by these molecules. [ABSTRACT FROM AUTHOR]

Objetivo: analizar y proponer un nuevo conjunto de SNPs para genotipificar MTB basado en secuencias genómicas obtenidas de América, África, Asia y Europa. Métodos: utilizamos 249 secuencias genómicas MTB obtenidas de las bases de datos mundiales: 125 reportaban genotipos conocidos determinados por el método de referencia (Spoligotyping), 76 reportaban genotipos conocidos determinados por análisis filogenético; y 48 no reportaban genotipos. Las secuencias se mapearon con el genoma de referencia H37Rv utilizando BWA y SAMtools, además se identificó SNPs utilizando kSNP y ParSNP. Las mutaciones en loci se transformaron a datos binarios y se determinaron mediante el criterio de inclusión/exclusión (IE) utilizando datos de Spoligotyping y filogenias, se excluyó el análisis de SNPs en regiones no secuenciadas; luego se seleccionó los SNPs no sinónimos presentes en clúster de grupo ortólogos (COG) más conservados de cada genotipo MTB. Finalmente, nuestro nuevo conjunto propuesto se analizó en 34 secuencias genómicas MTB adicionales con genotipos conocidos. Resultados: para desarrollar el sistema de asignación de SNPs y genotipos, primero utilizamos el criterio IE. En una base de datos, integramos 7649 SNPs encontrados a partir de 249 secuencias genómicas de MTB que incluyen los genotipos Beijing, CAS, EAI, Haarlem, LAM, X, Ural, AFRI1 y AFRI2. Posteriormente, se excluyeron los SNPs compartidos entre los diferentes genotipos. Finalmente, se seleccionó los SNPs no sinónimos presentes en el COG más conservado de cada genotipo de MTB. Se generó una base de datos de SNPs, que incluye SNPs, gen y COG, reportando 2, 36, 20, 4, 2, 2, 40, 65 y 67 SNPs para Beijing, CAS, EAI, Haarlem, LAM, X, Ural, AFRI1 y AFRI2 respectivamente. Seleccionamos 10 SNPs no sinónimos para 9 genotipos MTB analizados (Tabla 1) y validamos nuestro resultado utilizando 34 genomas MTB adicionales obteniendo una correlación del 100% con sus genotipos conocidos. Conclusiones: El conjunto de SNPs propuesto abarca los genotipos MTB más prevalentes a nivel mundial: Beijing, CAS, EAI, Haarlem, LAM, X, Ural, AFRI1 y AFRI2. Este conjunto de SNPs abre la posibilidad de realizar estudios de epidemiología molecular y marcadores de resistencia a drogas antituberculosas. [ABSTRACT FROM AUTHOR]

Objetivos: analizar la expresión de un perfil de microARNs para el diagnóstico diferencial de tuberculosis (TB) latente y TB activa. Métodos: se realizó un estudio retrospectivo. Un total de 30 muestras de suero fueron seleccionadas, de los cuales 10 fueron controles sanos, 10 pacientes con diagnóstico de TB latente y 10 pacientes con diagnóstico de TB activa. Se extrajo el ARN con kit comercial (miRNeasy, Qiagen), la síntesis de cDNA y posterior PCR en tiempo real se realizó utilizando kit comercial (TaqMan® MicroRNA Assays, Life Technologies). Se utilizó RNU48 como gen normalizador (housekeeping gene). La detección de Ct (threshold cycle) para cada microARN se realizó en el equipo Rotogene Q (Qiagen). Cada muestra se corrió por duplicado. El análisis de expresión diferencial de miR-21, miR-29a, miR-99b y miR-155 se realizó por el método de Ct comparativo o 2-ddCt (Método de Livak). Resultados: los resultados de cuantificación relativa por PCR en tiempo real muestran valores de Ct diferentes en cada curva de fluorescencia para los microARNs miR-21, miR-29a, miR-99b y miR-155 respecto al normalizador RNU48, los cuales nos dan un perfil de expresión diferencial respecto a controles sanos. El nivel de expresión de miR-21 fue mayor en pacientes con TB activa. No hay diferencias en la expresión diferencial de miR-99b en pacientes con TB latente y TB activa. Los microARNs miR-29a y miR-155 presentan mayor expresión diferencial en pacientes con TB latente. Conclusiones: miR-21 podría ser considerado como biomarcador para diagnóstico de TB activa, mientras que miR-29a y miR-155 podrían ser usados como biomarcadores para diagnóstico de TB latente. Es necesario la validación de estos microARNs en una población mayor de pacientes, así mismo explorar la expresión diferencial de otros microARNs específicos. [ABSTRACT FROM AUTHOR]

We report the use of a mobile laboratory set up to extract ancient DNA (aDNA) from 34 human coprolites (fossilized faeces) samples. Our approach enabled the rapid genetic characterization of 5,000 years old archeological samples. It is useful for the on-site screening of museums and freshly excavated samples for DNA. This approach is accessible to other investigators as the mobile laboratory was set up using commercially available instruments., (© 2022 Biomedical Informatics.)

Objectives: To analyze the differential expression of miR-21, miR-29a, miR-99b and miR-155 in serum samples from patients with latent tuberculosis (TB) and active TB compared to healthy controls., Mate Rials and Methods: We used 28 serum samples (9 with active TB, 10 with latent TB and 9 healthy con trols) for the analysis of gene expression by RT-qPCR with Primers and TaqMan probes. The differential expression was calculated by the Livak method using a normalizing gene (RNU-48)., Results: Overex pression of miR-155 was found in people with latent tuberculosis, compared to healthy controls (0.63 vs. 0.01; p value = 0.032)., Conclusion: The miR-155 could be considered a biomarker to differentiate latent TB from active disease. Studies with larger sample sizes are required to corroborate the findings.

The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains is a major health problem for high Tuberculosis (TB) incidence countries. Therefore, it is of interest to identify antibiotic resistant bacteria by mismatch detection using DNA hybridization. We generated PCR products for five genes (rpoB, inhA, katG, gyrA and rrs) associated with drug resistance TB from MDR and XDR Mycobacterium tuberculosis (MTB) DNA samples. These were hybridized to PCR products from MTB H37Rv (pansusceptible laboratory strain) to generate DNA hetero-duplex products, which was digested by Detection Enzyme (GeneArt Genomic Cleavage Detection Kit) and visualized by agarose gel electrophoresis. Results show different bands with sizes of 400 bp and 288 bp (rpoB), 280 bp (inhA), 310 bp (katG), 461 bp (gyrA) and 427 bp (rrs) suggesting mutations in DNA heteroduplex for each gene. Detection Enzyme specifically cleaves DNA hetero-duplex with mismatch. The technique helps in the improved detection of MDR (mutations in rpoB, inhA and katG) and XDR (mutations in rpoB, inhA katG, gyrA and rrs) MTB strains. Moreover, the technique is customized without expensive specialized equipment to detect mutations. It is also fast, efficient and easy to implement in standard molecular biology laboratories.

Objectives.: To analyze melting curves for the diagnosis of multidrug-resistant tuberculosis from sputum samples., Materials and Methods.: Sputum samples (n = 250) were collected from patients with clinical suspicion of pulmonary tuberculosis as a result of bacilloscopy and cultured in solid medium Lowenstein Jensen. According to the reference method, 124 samples sensitive to rifampicin and isoniazid, 24 resistant to rifampicin, 33 resistant to isoniazid, and 69 multidrug-resistant were used. It was evaluated by real-time PCR and then by melting curves, the rpoB gene was used as a biomarker of rifampicin resistance, and the katG gene and inhA promoter region were used as biomarkers of isoniazid resistance. The H37Rv strain was considered a drug-sensitive control. The results of the reference method and the results of the melting curve analysis were compared to evaluate the parameters of sensitivity, specificity, positive predictive value and negative predictive value., Results.: Rifampicin resistance showed a sensitivity of 90.3%, specificity of 90.4%, positive predictive value of 84.8% and negative predictive value of 94.0%. Isoniazid resistance showed a sensitivity of 90.2%, specificity of 93.9%, positive predictive value of 91.1% and negative predictive value of 93.3%. The detection of multidrug-resistant tuberculosis showed values of 89.9%, 90.6%, 78.5% and 95.9% for sensitivity, specificity, positive predictive value and negative predictive value, respectively., Conclusions.: The melting curve analysis showed to be safe and reliable to be used in the rapid diagnosis of multidrug-resistant tuberculosis in sputum samples.

Lung cancer is a leading cause of cancer-related death among both men and women in the United States, where non-small cell lung cancer accounts for ∼85% of lung cancer. Lung adenocarcinoma (ADC) is the major histologic subtype. The presence of actionable mutations prompts the use of therapies designed to specifically address the deleterious effects of those cancer-driving mutations; these therapies have already shown promise in cases carrying those actionable mutations (∼30%). Innovative therapeutic approaches are needed for the treatment of 70% of patients suffering from lung ADC. Adoptive transfer of CD8 + T cells specific against cancer/testis (CT) Ags, whose protein expression is restricted to the gonads (testis and ovary) and cancerous cells, is an excellent alternative. In this study, we report the isolation of HLA-A*02:01/CT37 peptide-specific α and β TCR chains from a CD8 + T cell clone obtained from a patient suffering from lung ADC. We also report the development of an innovative CD3ζ construct. With those TCR chains and the engineered (modified) CD3ζ chain, we produced a construct that when transduced into CD8 + T cells is capable of redirecting transduced CD8 + T cell cytotoxic activity and IFN-γ secretion against peptide-pulsed autologous cells and HLA-A*02:01 -positive and CT37-expressing lung ADC cell lines. Our findings will launch the development of innovative adoptive transfer immunotherapies for the treatment of lung ADC, targeting the most prevalent HLA molecules and CT37 peptides restricted by these molecules., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), has a vast diversity of genotypes including Beijing, CAS, EAI, Haarlem, LAM, X, Ural, T, AFRI1 and AFRI2. However, genotyping can be expensive, time consuming and in some cases, results may vary depending on methodology used. Here, we proposed a new set of 10 SNPs using a total of 249 MTB genomes, and selected by first the inclusion/ exclusion (IE) criteria using spoligotyping and phylogenies, followed by the selection of the nonsynonymous SNPs present in the most conserved cluster of orthologous groups (COG) of each genotype of MTB. Genotype assignment of the new set of 10 SNPs was validated using an additional of 34 MTB genomes and results showed 100% correlation with their known genotypes. Our set of 10 SNPs have not been previously reported and cover the MTB genotypes that are prevalent worldwide. This set of SNPs could be used for molecular epidemiology with drug resistant markers.

Tuberculosis is a health problem worldwide with one-third of the population infected with the Mycobacterium tuberculosis bacilli. The first-line of treatment for tuberculosis includes the drugs Isoniazid (INH) and Rifampicin (RIF) metabolized in the liver. Drug metabolism is directly related to the genetic variation of NAT2 and CYP2E1 (associated with INH metabolism) and AADAC (associated with RIF metabolism), and the effects produced in an individual may be a fast, intermediate or slow metobolizer. Polymorphisms in genes of people in standard tuberculosis treatment can cause effects on drug metabolism with consequences of hepatotoxicity and even drug resistance. Countries have began clinical trials focused on personalization of tuberculosis treatment to reduce the consequences for patients in treatment. In countries like Peru, where high rates of tuberculosis are recorded and therefore more people in treatment, the pharmacogenomic of individuals becomes a crucial tool for an optimum tuberculosis treatment. This review highlights the importance of having pharmacogenomic studies and having the identification of polymorphisms associated to the metabolism of the anti-tuberculosis drugs in our Peruvian population.

Based on the tuberculin skin test it is estimated that latent tuberculosis infection is present in one-third of the world's population. The new strategies in public health and research are aimed to reduce and eradicate this enormous reservoir. However, the absence of effective biomarkers for diagnosis and treatment of latent tuberculosis limits the development of new drugs and vaccines. Some components are present in both, the PPD (used in the tuberculin skin test) and the BCG vaccine. This increases the number of false positives in vaccinated individuals. Nowadays, there is not an immune diagnostic method that can differentiate latent tuberculosis and tuberculosis disease. New studies have addressed some strategies including specific antibodies, new cytokines and / or antigens as candidates for biomarkers. However, the high costs of these studies, the low number of participants and their different methodology make difficult a future meta-analysis and more conclusive results., (Copyright © 2010 Elsevier España, S.L. All rights reserved.)

Our objective was to determine the genotype of the dengue virus type 2 (DENV-2) that circulated in the Amazon region of Peru between November 2010 and January 2011. We analyzed eight samples collected during dengue surveillance activities in the cities of Iquitos, Yurimaguas, Trujillo, Tarapoto and Lima between November 2010 and January 2011 that were sent to Insitituto Nacional de Salud. The viruses were isolated in C6/36 HT cell line. Viral RNA was extracted and the serotype (RT - PCR multiplex) and genotype (RT-Nested PCR of the region E/NS1) were determined. Finally, the E/ NS1 amplicons were sequenced and analyzed by phylogeny. The phylogenetic analysis revealed the introduction of a different lineage which entered in Peru by the end of 2010. These isolates found in Iquitos and other cities in Peru are closely related to DENV-2 isolates that circulated in Brazil during 2007 and 2008, associated with severe dengue cases and deaths. In conclusion, we detected the introduction of a different lineage of DENV-2 America / Asia genotype in Peru that could be associated with the presence of more severe cases.