Detección y cuantificación de VHB y VHC en muestras de plasma mediante la utilización de diferentes ensayos moleculares: estudio comparativo / Detection and quantification of HBV and HCV in plasma samples by means of different molecular assays: Comparative study

Introducción: La carga viral es un marcador muy útil para realizar el seguimiento de los pacientes infectados por VHB y VHC. Este trabajo compara ensayos basados en amplificación mediada por transcripción y en PCR a tiempo real con el objetivo de comprobar si pueden ser intercambiables. Material y métodos: Estudio bicéntrico en el que se analizó la carga viral de 147 muestras de plasma de pacientes infectados por VHB y 229 por VHC, mediante ensayos basados en amplificación mediada por transcripción (Aptima® HBV Quant y Aptima® HCV Quant Dx, que utilizan el sistema Panther (Hologic®)) y PCR a tiempo real (COBAS® AmpliPrep / COBAS® TaqMan® y COBAS® 6800), calculando el grado de concordancia entre ellos. Resultados: Se detectó carga viral en ambos equipos en 60 (40,82%) muestras de VHB (mediana del log de la carga viral: COBAS®: 2,51UI/mL (RIC 2,20-3,17), Panther: 2,71UI/mL (RIC 2,21-3,22)) y en 39 (16,96%) muestras de VHC (mediana del log de la carga viral: COBAS®: 3,93UI/mL (RIC 2,24-6,01), Panther: 3,80UI/mL (RIC 1,99-6,14)). La concordancia entre ambos equipos fue de κ=0,943 para VHB y κ=0,925 para VHC. La comparación de las muestras con carga viral detectada mediante los 2 ensayos mostró una correlación alta tanto para VHB (R2=0,86) como para VHC (R2=0,97). Conclusiones: Los ensayos basados tanto en amplificación mediada por transcripción como en PCR a tiempo real pueden ser intercambiables para el manejo de pacientes infectados con VHB y VHC.(AU)

Introduction: Viral load is a very useful marker for monitoring patients infected with HBV and HCV. This work compares assays based on transcription-mediated amplification and on real-time PCR to verify whether they can be interchangeable. Material and methods: a bicentric study, in which 147 plasma samples from patients infected with HBV and 229 with HCV were analyzed, was carried out. Transcription-mediated amplification-based assays (Aptima® HBV Quant and Aptima® HCV Quant Dx, employing Panther system (Hologic®)) and on real-time PCR (COBAS® AmpliPrep / COBAS® TaqMan® and COBAS® 6800) were used and the degree of concordance between them was calculated. Results: Viral load was detected in both systems in 60 (40.82%) HBV samples (median log viral load: COBAS®: 2.51IU/mL (IQR 2.20-3.17), Panther: 2.71IU/mL (IQR 2.21-3.22)) and in 39 (16.96%) HCV samples (median log viral load: COBAS®: 3.93IU/mL (IQR 2.24-6.01), Panther: 3.80IU/mL (IQR 1.99-6.14)). The agreement between both systems was κ=0.943 for HBV and κ=0.925 for HCV. Comparison of viral load samples detected by both assays showed a hight correlation for HBV (R2=0.86) and for HCV (R2=0.97). Conclusions: Both transcription-mediated amplification and on real-time PCR based assays may be interchangeable for the management of patients infected with HBV and HCV.(AU)

Detection and quantification of HBV and HCV in plasma samples by means of different molecular assays: Comparative study.

INTRODUCTION: Viral load is a very useful marker for monitoring patients infected with HBV and HCV. This work compares assays based on transcription-mediated amplification (TMA) and on real-time PCR (RT-PCR) to verify whether they can be interchangeable. MATERIAL AND METHODS: A bicentric study, in which 147 plasma samples from patients infected with HBV and 229 with HCV were analyzed, was carried out. TMA-based assays (Aptima® HBV Quant and Aptima® HCV Quant Dx, employing Panther system (Hologic®)) and RT-PCR (COBAS® AmpliPrep/COBAS® TaqMan® and COBAS® 6800) were used and the degree of concordance between them was calculated. RESULTS: Viral load was detected in both systems in 60 (40.82%) HBV samples (median log viral load: COBAS: 2.51IU/mL (IQR 2.20-3.17), Panther: 2.71IU/mL (IQR 2.21-3.22)) and in 39 (16.96%) HCV samples (median log viral load: COBAS: 3.93IU/mL (IQR 2.24-6.01), Panther: 3.80IU/mL (IQR 1.99-6.14)). The agreement between both systems was κ=0.943 for HBV and κ=0.925 for HCV. Comparison of viral load samples detected by both assays showed a hight correlation for HBV (R2=0.86) and for HCV (R2=0.97). CONCLUSIONS: Both TMA and RT-PCR based assays may be interchangeable for the management of patients infected with HBV and HCV.

Ribavirin as a First Treatment Approach for Hepatitis E Virus Infection in Transplant Recipient Patients.

The hepatitis E virus (HEV) is the major cause of acute hepatitis of viral origin worldwide. Despite its usual course as an asymptomatic self-limited hepatitis, there are highly susceptible populations, such as those with underlying immunosuppression, which could develop chronic hepatitis. In this situation, implementation of therapy is mandatory in the sense to facilitate viral clearance. Currently, there are no specific drugs approved for HEV infection, but ribavirin (RBV), the drug of choice, is used for off-label treatment. Here, we present two cases of chronic HEV infection in transplant patients, reviewing and discussing the therapeutic approach available in the literature. The use of RBV for the treatment of an HEV infection in organ transplant patients seems to be effective. The recommendation of 12 weeks of therapy is adequate in terms of efficacy. Nevertheless, there are important issues that urgently need to be assessed, such as optimal duration of therapy and drug dosage.

Actualización de la labor de los Comités de Ética de la Investigación en la evaluación de la investigación genética y en su función como comité de ética externo de un biobanco / Update of the work of the Ethics Research in evaluating genetic research and its role as an external ethics committee biobank

Las investigaciones sobre el genoma humano y sus aplicaciones abren inmensas perspectivas de mejora en la salud de los individuos. Sin embargo, estos avances no deben poner nunca en riesgo el respecto por la dignidad, la libertad y los derechos de los participantes en la investigación, garantizando la prohibición de toda forma de discriminación fundada en las características genéticas. Los Comités de Ética de la Investigación ( CEI), responsables de velar por la protección de los derechos, seguridad y bienestar de los sujetos que participar en la Investigación Biomédica, evalúan estudios genéticos presentados de forma independiente, ensayos clínicos en los que el objetivo principal es la obtención de información genética y subestudios genéticos de ensayos clínicos con medicamentos (…) (AU)

Research on human genome and its applications open great perspectives to improve human being´s health. However, these advances must never endanger the respect of dignity, freedom and rights of the participants in medical research, assuring prohibition of any way of discrimination because of genetic features. The Independent Research Boards (IRB), responsible for safeguarding rights, safety and well-being of the subjects taking part in the biomedical research, assess independently submitted genetic studies, clinical trials whose primary objective is obtaining genetic information and genetic sub-studies of clinical trials with drugs (…) (AU)

Uso rutinario del Pneumonia Severity Index en el servicio de urgencias: efecto sobre los indicadores de proceso y resultado en neumonía adquirida en la comunidad / The routine use of the Pneumonia Severity Index in the emergency department: Effect on process-of-care indicators and results in community acquired pneumonia

Objetivos Evaluación de indicadores de proceso (hospitalización inadecuada, adecuación y precocidad de antibioterapia) y resultado (estancia hospitalaria, reingresos, ingresos UCI y mortalidad) de neumonía adquirida en la comunidad (NAC) al aplicar la guía SEPAR/IDSA. Pacientes y métodos Estudio observacional retrospectivo de pacientes consecutivos con diagnóstico al alta de NAC en los primeros semestres de 2007 y 2008 (186 y 161 pacientes, respectivamente) atendidos en urgencias de hospital general. Se han analizado las diferencias en los indicadores entre los grupos de pacientes con/sin Pneumonia Severity Index (PSI) calculado, según el año de implantación de la guía, y se compararon con los de 110 pacientes de 2006 según la guía SEQ/ATS. Resultados La guía SEPAR/IDSA ha mejorado los indicadores de proceso: mayor adecuación del ámbito de tratamiento, disminución de ingresos injustificados (del 39,4% en 2006 al 8,5% en 2007 [p<0,001] y al 17,2% en 2008 [p=0,005]) y mayor precocidad de antibioterapia. No se han modificado los indicadores de resultado. En 2008 se observó una reducción de la mortalidad en el subgrupo de pacientes con PSI IV-V en los que se calculó el PSI (2,3%), respecto a los que no se calculó (28,3%; p<0,001), y esta tasa fue inferior a la de los pacientes con criterios de hospitalización según la guía SEQ/ATS (22,7%; p=0,003).Conclusión La guía SEPAR/IDSA ha reducido los ingresos injustificados y en el segundo año de aplicación se ha observado mayor precocidad de la antibioterapia junto a una reducción de la mortalidad en los pacientes con riesgo moderado-alto en los que se calculó el PSI (AU)

Objective To evaluate process-of-care indicators (inappropriate hospitalisation, suitability and early antibiotic treatment) and outcome indicators (length of hospital stay, hospital readmission, ICU admission, and mortality) in the management of community-acquired pneumonia (CAP) when the SEPAR/IDSA guidelines were applied. Patients and methods An observational retrospective study conducted on patients diagnosed with CAP during the first semester of 2007 and 2008 (186 and 161 patients, respectively) in the emergency unit of a general hospital. Differences in the process-of-care and outcome indicators between 2007 and 2008 (with and without the Pneumonia Severity Index [PSI]) were evaluated. Moreover, the indicators were compared with those obtained in 2006 (110 patients), when the current guidelines were those of SEQ/ATS. Results The SEPAR/IDSA guidelines improved the following process-of-care indicators: appropriateness of treatment, unjustified hospital readmission (39.4% in 2006 vs. 8.5% in 2007 [P<.001], and 17,2% in 2008 [P=.005]), and early treatment. However, outcome indicators did not change. In 2008, a decrease in the mortality of the patients of risk classes IV-V in which the PSI had been estimated was observed in comparison with the patients in which the PSI was not estimated (2.3% vs. 28.3%; P<.001). Moreover, the mortality rate of the patients of risk classes IV-V in which the PSI had been estimated was lower than those measured using the SEQ/ATS guidelines (22.7%; P=.003).Conclusion SEPAR/IDSA guidelines decreased the unjustified hospital readmission. In the second year of its application an increase in the number of patients who received early treatment, and a decrease of the mortality rate of the patients of risk classes IV-V in which the PSI had been estimated, were also observed (AU)

Quantitative determination of the okadaic acid toxins group by a colorimetric phosphatase inhibition assay: interlaboratory study.

An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

[Update of the work of the ethics research in evaluating genetic research and its role as an external ethics committee biobank]. / Actualización de la labor de los Comités de Etica de la Investigación en la evaluación de la investigación genética y en su función como comité de ética externo de un biobanco.

Research on human genome and its applications open great perspectives to improve human beings' health. However, these advances must never endanger the respect of dignity, freedom and rights of the participants in medical research, assuring prohibition of any way of discrimination because of genetic features. The Independent Research Boards (IRB), responsible for safeguarding rights, safety and well-being of the subjects taking part in the biomedical research, assess independently submitted genetic studies, clinical trials whose primary objective is obtaining genetic information and genetic sub-studies of clinical trials with drugs. Biobanks, as safeguarding means to preserve biological samples in suitable quality conditions, must be assigned to two external committees, a scientific one and an ethics one. External ethics committees of biobanks have to make the ethical assessment of the submissions of samples transfers and associated data, in order to carry out research projects. On the other hand, they have to advise biobanks on the compliance of ethical and legal principles, which, in many committees, has turned into the performance of informed consent forms which are in accordance with current laws.

[The routine use of the Pneumonia Severity Index in the emergency department: effect on process-of-care indicators and results in community acquired pneumonia]. / Uso rutinario del Pneumonia Severity Index en el servicio de urgencias: efecto sobre los indicadores de proceso y resultado en neumonía adquirida en la comunidad.

OBJECTIVE: To evaluate process-of-care indicators (inappropriate hospitalisation, suitability and early antibiotic treatment) and outcome indicators (length of hospital stay, hospital readmission, ICU admission, and mortality) in the management of community-acquired pneumonia (CAP) when the SEPAR/IDSA guidelines were applied. PATIENTS AND METHODS: An observational retrospective study conducted on patients diagnosed with CAP during the first semester of 2007 and 2008 (186 and 161 patients, respectively) in the emergency unit of a general hospital. Differences in the process-of-care and outcome indicators between 2007 and 2008 (with and without the Pneumonia Severity Index [PSI]) were evaluated. Moreover, the indicators were compared with those obtained in 2006 (110 patients), when the current guidelines were those of SEQ/ATS. RESULTS: The SEPAR/IDSA guidelines improved the following process-of-care indicators: appropriateness of treatment, unjustified hospital readmission (39.4% in 2006 vs. 8.5% in 2007 [P<.001], and 17,2% in 2008 [P=.005]), and early treatment. However, outcome indicators did not change. In 2008, a decrease in the mortality of the patients of risk classes IV-V in which the PSI had been estimated was observed in comparison with the patients in which the PSI was not estimated (2.3% vs. 28.3%; P<.001). Moreover, the mortality rate of the patients of risk classes IV-V in which the PSI had been estimated was lower than those measured using the SEQ/ATS guidelines (22.7%; P=.003). CONCLUSION: SEPAR/IDSA guidelines decreased the unjustified hospital readmission. In the second year of its application an increase in the number of patients who received early treatment, and a decrease of the mortality rate of the patients of risk classes IV-V in which the PSI had been estimated, were also observed.

Extension of the validation of AOAC Official Method 2005.06 for dc-GTX2,3: interlaboratory study.

AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.

Determination of lipophilic toxins by LC/MS/MS: single-laboratory validation.

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.

High-throughput analysis of amnesic shellfish poisoning toxins in shellfish by ultra-performance rapid resolution LC-MS/MS.

The application of ultra-performance rapid resolution LC on a 1.8 microm particle-size column coupled with tandem MS (RRLC-MS/MS) is described for the analysis of amnesic shellfish poisoning (ASP) toxins in shellfish. Complete resolution among domoic acid (DA) and the isomers was achieved in less than 3 min. The method was intralaboratory validated for direct analysis of crude extracts without further cleanup. It showed LODs ranging from 0.05 to 0.09 mg/kg and a working range that complied with the current regulatory level for DA of 20 mg/kg, and with the level of 4.5 mg/kg recently proposed by the European Food Safety Authority. Confirmatory capabilities were demonstrated according to the Commission Decision 2002/657/EC criteria. The results obtained by RRLC-MS/MS agreed with those provided by the reference LC-UV method, both intralaboratory for the analysis of blind samples (R2 = 0.9751) and interlaboratory through participation in the proficiency test for ASP toxins during 2009 (z-score = -0.962 and 0.177 for low- and high-contaminated samples, respectively). RRLC-MS/MS provided fast analysis and additional confirmatory capabilities for direct analysis of crude extracts while the performance and reliability of the results were maintained, even in very complex matrixes.