Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow.

BACKGROUND: In recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking. METHODS: A total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7). RESULTS: The optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses). CONCLUSIONS: Although the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

Labor and neonatal outcomes after term induction of labor in gestational diabetes.

OBJECTIVE: To identify the optimal gestational age (GA) for induction of labor (IOL) at term among patients with gestational diabetes (GDMA) according to perinatal outcomes. STUDY DESIGN: The US Natality Database from 2007 to 2010 was reviewed. Inclusion criteria were singleton delivery, IOL at 37 to 42 weeks and GDMA. Exclusion criteria included congenital anomalies, pre-gestational diabetes, hypertensive disorders, previous cesarean, breech presentation and rupture of membranes. Controls were non-GDMA cases delivered in geographic and temporal proximity. Delivery mode, macrosomia and perinatal complications were analyzed. Logistic regression adjusted for confounders was used to calculate odds ratios by GA using 39 weeks non-GDMA as reference. RESULTS: In all, 96,964 cases and 176,079 controls were included. Increased risk for all adverse outcomes among GDMA cases was found. The nadir for intrapartum and neonatal complications was 38 and 40 weeks, respectively, whereas for cesarean and macrosomia was 39 weeks. CONCLUSION: The optimal timing for IOL at term in GDMA appears to be 39 to 40 weeks.

Endometriosis pélvica-linfática simulando: cáncer de ovario avanzado / Pelvic-lymphatic endometriosis simulating: neoplasms of advanced ovary

La endometriosis es una enfermedad que afecta a la mujer en edad fértil. Consiste en tejido endometrial fuera del abdomen. Los estimados actuales indican que fluctúa entre el 2 y el 10 por ciento en la edad reproductiva. Alrededor de un 30 a 40 por ciento de las mujeres con endometriosis no son fértiles, siendo una de las causas de infertilidad femenina. La endometriosis ganglionar es un fenómeno infrecuente. Más raro aún es hallar Endometriosis Linfática - Ganglionar simulando un cáncer de ovario avanzado.

Aislamiento de plásmidos conjugativos en cepas de enterobacterias productoras de ß-lactamasas de espectro expandido / Isolation of conjugative plasmids in enterobacteriaceae strains producing beta-lactamases expanded sprectrum

Las ß-lactamasas de espectro expandido son enzimas capaces de hidrolizar el enlace amida de los oximino ß-lactámicos, (cefotaxime, ceflazidime y aztreonam). Las mismas son codificadas en plásmidos y por lo tanto pueden ser transferidas mediante conjugación a diversos géneros bacterianos. Diseminándose ampliamente en el ambiente hospitalario. En esta investigación se persigue, en cepas de enterobacterias aislar plásmidos que codifican para ß-lactamasas de espectro expandido y que sean capaces de transferirse mediante conjugación. Se estudio una población de 51 enterobacterias productoras de ß-lactamasas de espectro expandido aisladas de diferentes centros hospitalarios del área Metropolitana de Caracas. A las mismas se le determinó el perfil de resistencia a múltiples antibióticos mediante la metodología de Kirby-Bauer. Se detectaron las BLEE mediante dos ensayos fenotípicos basados en el efecto sinergístico con el ácido clavulánico y se tipificaron molecularmente por PCRIRFLP, seguidamente se transfirieron los plásmidos conjugativos en ensayos de conjugación en medio sólido y se aislaron los plásmidos de cepas donantes y transconjugautes, por el método de lisis alcalina. Los resultados fenotípicos indican una mayor proporción de BLEE con actividad ceflazidimasa y en menor grado actividad cefotaximasa. La tipificación molecular indicó que 60,8 por ciento de las cepas portan genes tipo SHV y 15,6 por ciento codifican ß-lactamasas de espectro expandido de la familia CTX-M. De 36 cepas conjugadas un 81 por ciento transfirió material plasmídico. El análisis de los aislamientos plasmídicos mostró la presencia en la totalidad de las transcojugantes de una banda de 25000 pb y en un 80 por ciento se evidenció una banda plasmídica mayor a 50000 pb. Se pudo constatar la cotransferencia de resistencia a otras familias de antibióticos.

How bacteria protect themselves against channel-forming colicins.

Here we review the mechanisms that bacterial cells use to protect themselves against channel-forming colicins. Four mechanisms are examined: immunity, resistance, tolerance and PacB character. Immunity confers protection to colicinogenic cells against the colicin they produce, since the colicinogenic plasmid bears the genetic determinant for such immunity protein. Resistance is provided by modifications on colicin receptors located on the outer membrane. It prevents colicin adsorption and protects against those colicins sharing a common receptor. Tolerance is achieved by changes in the translocation system. The adsorbed colicin is not translocated toward the periplasmic space. This impedes its insertion into the cell membrane as well as the formation of the transmembrane channel. Tolerance confers protection against colicins that share the same translocation system. Finally, we discuss the PacB character, that confers protection against all known channel-forming colicins. The latter property is encoded by non-colicinogenic plasmids in the H-incompatibility complex.

Cloning of the PacB-Ter region from plasmid Mip233 (IncHI3) and their expression in E. coli ton, tol mutants.

A region of the plasmid Mip233 (incompatibility group HI3) encoding the phenotypes of resistance to the channel-forming colicins (character PacB) and potassium tellurite (Ter), was cloned and studied. Both properties are contained in an insert of 2.2 Kbp, being the smallest functional clone (pB22) isolated so far. E. coli DH5 alpha pB22 transformants exhibit resistance to the colicins as well as to high levels of tellurite (> 1000 micrograms ml-1). Results suggest that they are genetically linked forming an inducible operon. pB22 does not show significant homology with DNA from other H plasmids. Tests using E. coli ton and tol mutants harbouring recombinant pB22 indicate that the product of gene tolC, but not that of tonB, is required for the expression of the PacB and Ter phenotypes.

Presencia del Dermatoxys veligera Rudolphi, 1819) en conejo de monte (Sylvillagus floridanus) del estado Zulia / Presence of Dermatoxys veligera (Rudolphi, 1819) in rabbit-root (Sylvilagus floridanus) in Zulia State

En este trabajo de investigación, se identifica por primera vez para el Estado Zulia (Venezuela), la presencia del Dermatoxys veligera, Rudolphi, 1819, en el conejo de monte (Sylvilagus floridanus)