A Sigmoidal Model for the Interpretation of Quantitative PCR (QPCR) Experiments

Real-time or quantitative PCR (qPCR) is the most commonly used technique for estimating the amount of starting nucleic acids in a PCR or RT-PCR reaction. Quantification of PCR product is achieved in real time by measuring the increase in fluorescence of intercalating dyes, labeled primers or oligonucleotides in the presence of double stranded DNA. This amplification curve follows a sigmoid behavior and is used to estimate the relative and/or absolute amount of template using different methods and assumptions. Estimation of C0 normally requires the measurement of a threshold cycle and some assumption about the efficiency of the reaction. An accurate estimation of efficiency is paramount for a precise determination of template levels at time zero. Several non-linear fitting methods have been implemented to model the sigmoid behavior using different empirical models with varying amounts of parameters; however, interpretation of the corresponding parameters is not straightforward. In this paper a model of PCR amplification is deduced and used in the interpretation of qPCR experiments. A non-linear regression analysis of this equation gives a direct estimation of C0 and automatically calculates a parameter k related to the reaction efficiency. This model takes into account non-idealities in the amplification reaction and avoids a priori assumptions about efficiency

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Identificación serológica y molecular de los agentes causales asociados a enfermedades virales del tomate de árbol (solanum betaceum) en cultivos del municipio de Córdoba (Nariño)

Las enfermedades ocasionadas por agentes virales que afectan a las solanáceas se han convertido en un factor limitante y perjudicial para los cultivos de tomate de árbol presentes en las regiones cultivadoras de Nariño. Estudios previos apuntan a un posible potyvirus y a diferentes virus isométricos como los agentes causales de estas enfermedades. No obstante la información que se tenía de la problemática era insuficiente puesto que las identidades taxonómicas de los virus asociados a la infección no se habían esclarecido completamente. Dicha situación fue abordada en este estudio mediante la evaluación de la incidencia de potyvirus y de otros virus asociados a este cultivo, utilizando pruebas de ELISA a partir de 130 muestras de cuatro cultivos afectados en el municipio de Córdoba, evaluándose también muestras provenientes de semilla sexual. Adicionalmente se determinó la identidad taxonómica del potyvirus y de un polerovirus, a partir de la secuenciación de la región de la cápside viral (CP) de diferentes aislamientos. Los resultados indicaron la presencia de potyvirus en todas las muestras analizadas, y además se determinó que los virus CMV (30.7%), ToMV (54%) y PLRV (70%) presentaban una alta incidencia en los cultivos de la región. El análisis molecular permitió identificar al potyvirus como miembro de la especie PVY, compartiendo niveles de identidad superiores al 92% con cepas obtenidas en cultivos de papa de diferentes países; no obstante el análisis filogenético evidenció la separación de los aislamientos provenientes de Córdoba, agrupándolos en un clado independiente, sin una clara afinidad con ninguna raza de PVY, por lo cual se requiere la secuenciación de regiones más variables del genoma, de manera que sea posible establecer su identidad intraespecífica. Las secuencias CP del polerovirus tuvieron identidades superiores al 99% con cepas de PLRV de papa en diferentes regiones del mundo. De gran interés resultó el hecho de detectar mediante ELISA al virus PLRV en material de semilla sexual de tomate de árbol, siendo necesario evaluar con más detalle dicha situación, dadas las importantes consideraciones epidemiológicas que supondría la transmisión de PLRV a través de este medio. Esta investigación revela la ocurrencia de un complejo viral asociado a la virosis del tomate de árbol en el sur de Colombia y con base en los análisis de secuencias plantea la posible infección cruzada de cepas de PVY y PLRV entre los cultivos de papa y tomate de árbol de la región, lo cual requiere confirmación experimental mediante evaluaciones de patogenicidad cruzada

A Sigmoidal Model for the Interpretation of Quantitative PCR (QPCR) Experiments

Real-time or quantitative PCR (qPCR) is the most commonly used technique for estimating the amount of starting nucleic acids in a PCR or RT-PCR reaction. Quantification of PCR product is achieved in real time by measuring the increase in fluorescence of intercalating dyes, labeled primers or oligonucleotides in the presence of double stranded DNA. This amplification curve follows a sigmoid behavior and is used to estimate the relative and/or absolute amount of template using different methods and assumptions. Estimation of C0 normally requires the measurement of a threshold cycle and some assumption about the efficiency of the reaction. An accurate estimation of efficiency is paramount for a precise determination of template levels at time zero. Several non-linear fitting methods have been implemented to model the sigmoid behavior using different empirical models with varying amounts of parameters; however, interpretation of the corresponding parameters is not straightforward. In this paper a model of PCR amplification is deduced and used in the interpretation of qPCR experiments. A non-linear regression analysis of this equation gives a direct estimation of C0 and automatically calculates a parameter k related to the reaction efficiency. This model takes into account non-idealities in the amplification reaction and avoids a priori assumptions about efficiency

Identificación serológica y molecular de virus del tomate de árbol (Solanum betaceum) en cultivos de Córdoba (Nariño, Colombia)

Introduction. The diseases caused by viruses have become an obstacle for tree tomato crops in several Colombian regions, including those from the Nariño province. Previous studies pointed at a possible potyvirus and at several isometric viruses as associated to these diseases, though their taxonomic identity in the species level and their incidence had not been determined yet. An approach to this situation was made by the use of ELISA, RT-PCR and viral sequence capsid tests from samples brought from five crops affected by viruses in Córdoba (Nariño). Results. The results indicated the presence of potyvirus in all of the samples analyzed, while CMV virus (33%), ToMV (50%) and PLRV (75%) had a great incidence in the crops from the region.The molecular analysis allowed the identification of potyvirus as a member of the PVY species, sharing identity levels above 92% with strains obtained in potato crops from several countries, while the polerovirus sequences obtained identities above 98% with PLRV strains from the same crop. Conclusion. This research work confirms that there is a viral complex associated to viruses in tree tomatoes from the Nariño province and, base on the sequence analysis, exposes the possible crossed infection of PVY and PLRV strains in potato and tomato plantations in the region, a fact which requires an experimental confirmation by pathogenicity evaluations.Introducción. Las enfermedades causadas por virus se han convertido en un factor limitante para los cultivos de tomate de árbol presentes en diferentes regiones de Colombia, incluyendo aquellos del departamento de Nariño. Estudios previos apuntan a un posible potyvirus y a diferentes virus isométricos como asociados a estas enfermedades, aunque su identidad taxonómica en el nivel de especie y su incidencia no se habían determinado. Dicha situación fue abordada en este estudio utilizando pruebas de ELISA, RT-PCR y secuenciación de la cápside viral a partir de muestras de cinco cultivos afectados por la virosis en el municipio de Córdoba (Nariño). Resultados. Los resultados indicaron la presencia de potyvirus en todas las muestras analizadas, mientras que los virus CMV (33%), ToMV (50%) y PLRV (75%) presentaban una alta incidencia en los cultivos de la región. El análisis molecular permitió identificar al potyvirus como miembro de la especie PVY, compartiendo niveles de identidad superiores al 92% con cepas obtenidas en cultivos de papa de diferentes países; mientras que las secuencias del polerovirus presentaron identidades superiores al 98% con cepas de PLRV de este mismo cultivo. Conclusión. Esta investigación confirma la ocurrencia de un complejo viral asociado a la virosis del tomate de árbol en el departamento de Nariño, y con base en los análisis de secuencias, plantea la posible infección cruzada de cepas de PVY y PLRV entre los cultivos de papa y tomate de árbol de la región, lo cual requiere confirmación experimental mediante evaluaciones de patogenicidad

Sobreexpresión de genes en la interacción de Spongospora subterranea (Wallr.) Lagerh. y dos cultivares de Solanum phureja Juz. et. Buk

Con el fin de contribuir a un mejor conocimiento de los mecanismos que rigen la interacción entre Solanum phureja Juz. et. Buk. y Spongospora subterranea (Wallr.) Lagerh, agente causal de la sarna polvosa de la papa, se realizó en esta investigación el análisis del transcriptoma de dos cultivares: Criolla Colombia (susceptible) y Criolla Latina (tolerante), mediante pirosecuenciación 454. Los resultados indicaron diferencias entre los genes sobreexpresados en cada cultivar ante la infección con el patógeno. En el cultivar susceptible, la respuesta ocurre a partir de la activación transcripcional de genes asociados a la integridad de la pared celular, transducción de señales y genes involucrados en la respuesta a diferentes tipos de estrés. Los genes que codifican para metalotioneína (3297 veces), fosfatasa 2C (2128 veces) y un inhibidor de pectinmetilesterasa (2127 veces) fueron los más sobreexpresados en este cultivar. Por su parte, en el cultivar tolerante los niveles de sobreexpresión de genes fueron moderados, con sólo dos genes (un gen putativo y α-Galactosidasa) siendo transcritos más de 1000 veces, con respecto a las plantas sin inocular. Estos resultados sugieren que la tolerancia a S. subterranea de este cultivar, incluye también mecanismos de defensa constitutivos. Finalmente, mediante ensayos de qRT-PCR se confirmó la sobreexpresión de tres de los genes identificados en el transcriptoma del cultivar Criolla Latina.Palabras clave: NGS, PCR en tiempo real, sarna polvosa, transcriptoma.GENE OVEREXPRESSION IN TWO CULTIVARS OF SOLANUM PHUREJA JUZ. ET. BUK. IN INTERACTION WITH SPONGOSPORA SUBTERRANEAN (WALLR.) LAGERHABSTRACT: For a better understanding of the molecular mechanisms involved in the interaction between Solanum phureja Juz. et. Buk. and Spongospora subterranean (Wallr.) Lagerh, the causal agent of powdery scab disease, a 454 transcriptome analysis was carried out using one susceptible (Criolla Colombia) and one tolerant (Criolla Latina) potato cultivars. The results revealed a marked difference in the overexpression pattern for each cultivar. In the susceptible cultivar, the response was based on the activation of genes associated with cell-wall integrity, signal transduction and stress-related genes. The most overexpressed genes were those coding for methalothionein (3297 times), phosphatase 2C (2128 times) and a pectin methylesterase inhibitor (2127 times). Overexpression in the tolerant cultivar was moderate with only two genes (a putative gene and á-Galactosidase) being transcribed more than 1000 times compared with the non-inoculated control suggesting an important constitutive defense response. Overexpression of three genes in the Criolla Latina transcriptome was confirmed by qRT-PCR.Key words: NGS, powdery scab, real time PCR, transcriptome

Prosthetic Valve Candida spp. Endocarditis: New Insights Into Long-term Prognosis—The ESCAPE Study

International audienceBackground: Prosthetic valve endocarditis caused by Candida spp. (PVE-C) is rare and devastating, with international guidelines based on expert recommendations supporting the combination of surgery and subsequent azole treatment.Methods: We retrospectively analyzed PVE-C cases collected in Spain and France between 2001 and 2015, with a focus on management and outcome.Results: Forty-six cases were followed up for a median of 9 months. Twenty-two patients (48%) had a history of endocarditis, 30 cases (65%) were nosocomial or healthcare related, and 9 (20%) patients were intravenous drug users. "Induction" therapy consisted mainly of liposomal amphotericin B (L-amB)-based (n = 21) or echinocandin-based therapy (n = 13). Overall, 19 patients (41%) were operated on. Patients <66 years old and without cardiac failure were more likely to undergo cardiac surgery (adjusted odds ratios [aORs], 6.80 [95% confidence interval [CI], 1.59-29.13] and 10.92 [1.15-104.06], respectively). Surgery was not associated with better survival rates at 6 months. Patients who received L-amB alone had a better 6-month survival rate than those who received an echinocandin alone (aOR, 13.52; 95% CI, 1.03-838.10). "Maintenance" fluconazole therapy, prescribed in 21 patients for a median duration of 13 months (range, 2-84 months), led to minor adverse effects.Conclusion: L-amB induction treatment improves survival in patients with PVE-C. Medical treatment followed by long-term maintenance fluconazole may be the best treatment option for frail patients

Role of age and comorbidities in mortality of patients with infective endocarditis.

The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups: A total of 3120 patients with IE (1327  There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in th