A next-generation sequencing approach for the detection of mixed species in canned tuna.

Tuna cans are relevant seafood products for which mixtures of different tuna species are not allowed according to European regulations. In order to support the prevention of food fraud and mislabelling, a next-generation sequencing methodology based on mitochondrial cytochrome b and control region markers has been tested. Analyses of defined mixtures of DNA, fresh tissue and canned tissue revealed a qualitative and, to some extent, semiquantitative identification of tuna species. While the choice of the bioinformatic pipeline had no influence in the results (p = 0.71), quantitative differences occurred depending on the treatment of the sample, marker, species, and mixture (p < 0.01). The results revealed that matrix-specific calibrators or normalization models should also be used in NGS. The method represents an important step towards a semiquantitative method for routine control of this analytically challenging food matrix. Tests of commercial samples uncovered mixed species in some cans, being not in compliance with EU regulations.

The Influence of DNA Extraction Methods on Species Identification Results of Seafood Products.

In terms of species identification, the ultimate aim of extracting DNA is the subsequent amplification of the selected marker; therefore, the quality and quantity of the extracted DNA must be sufficient for PCR-based methods. The purpose of this study is to compare five DNA extraction methods according to the parameters of quantity, quality and simplicity, among others, in order to determine the most suitable method for identification for Cephalopoda, Gadiformes and Pleuronectiformes. The Wizard DNA clean-up system kit (Promega), MPure-12TM automated nucleic acid purification system (MP Biomedicals), Chelex 100 resin (Biorad), DNeasy blood and tissue kit (Qiagen) and a swab method were examined. The obtained DNA quantity was determined by fluorescence, and quality was evaluated with ratios of absorbance of A260/A280 and A260/A230 by agarose gel visualization of the extracts and by analyzing the success of PCR amplifications of 720 bp fragments of cytochrome c oxidase I (COI) for Cephalopods and 465 bp fragments of cytochrome b for Gadiformes and Pleuronectiformes. Statistical results confirmed significant differences between the tested methods according to yield, efficiency and purity and no significant differences with respect to the species employed. The best yields were obtained with the Wizard kit, whereas other methods stand out in terms of their affordability (Chelex) and automation (Mpure).

Improvement in RAID questionnaire results in patients with rheumatoid arthritis treated with advanced therapies.

OBJECTIVES: To analyse the changes in patient-reported outcomes after starting advanced antirheumatic treatment. METHODS: The study included all patients who started self-administered biological or targeted synthetic treatments for rheumatoid arthritis between February and November 2020. The patients were given the RAID quality of life questionnaire to complete before starting the treatment and after 4 months. Univariate and multivariate analyses were performed to determine the association between patients' clinical and sociodemographic characteristics and quality of life improvement. The level of significance was set at 0.05. RESULTS: Forty-six patients were included. Their ratings in the RAID questionnaire were improved after 4 months of treatment, both in the final overall total, which improved by 1.63±2.29 points, and in the different subtopics of the questionnaire (range 0-10). Pain was the domain that improved the most (2.33±2.82 points), followed by functional disability (2.15±2.51) and physical well-being (1.96±3.18). The improvement was statistically significant in all domains except the sleep score, which showed no statistically significant difference between the two time points analysed. CONCLUSIONS: Advanced antirheumatic treatment improves the quality of life of patients after 4 months of treatment.

Implementation of pharmaceutical care through Telepharmacy: A guide for professionals and patients.

OBJECTIVE: To provide a practical guide for the implementation and use of  Pharmaceutical Care through Telepharmacy by healthcare professionals and  patients in its different scopes of application. To establish a definition of  Telepharmacy and describe the technological tools necessary, advantages, and  keys to facilitating its implementation. METHOD: Between December 2020 and January 2021, the Board of Directors of the Spanish Society of Hospital Pharmacy, along with the coordinators of the  project "Outpatient Care Strategies" ("Mapa Estratégico para la Atención al  Paciente Externo") designed a strategy to foster the development and  expansion of Telepharmacy in Spain. This strategy involved four courses of  action. To develop the first course of action, a call was made in March 2021  among the Spanish Society of Hospital Pharmacy members to develop seven  methodological guidelines. The purpose of these documents was to meet the  needs for the implementation of Telepharmacy, the most relevant being the  development of specific guides for professionals and patients. The guides were  developed in four stages between May and October 2021, including a literature review; consensus-based interviews, online workshops, and, finally, the  drafting and validation of the final documents. Once the final draft was  prepared, a public evaluation of suggestions and observations was performed  for a month. The documents were also presented to the Patient Committee of  the Spanish Society of Hospital Pharmacy. RESULTS: The Guide for Professionals provides guidelines for the development and implementation of Telepharmacy in its different scopes of  application. These guides provide a description of specific goals, healthcare benefits, tools required, and keys to the implementation of  Telepharmacy. The Guide for Patients is complementary to face-to-face  pharmaceutical care from the point of view of the patient, with special  emphasis being placed on the most frequently used tools, the potential  benefits, and the keys to facilitating patients' understanding of the purpose  and use of Telepharmacy. CONCLUSIONS: A Practical Guide for Professionals and Patients was developed to ensure the standard development, implementation, and spread of Telepharmacy in all its scopes. This guide is intended to help Hospital Pharmacy professionals benefit from Telepharmacy as a  complementary tool to face-to-face pharmaceutical care.

OBJETIVO: Desarrollar una guía práctica sobre atención farmacéutica mediante  Telefarmacia para profesionales y pacientes, en cada uno de sus ámbitos de  aplicación. Definir en qué consiste, las herramientas utilizadas, los potenciales  beneficios y las claves para facilitar su comprensión.Método: Entre diciembre de 2020 y enero de 2021, la Junta directiva de la  Sociedad Española de Farmacia Hospitalaria, junto con coordinadores del  proyecto "Mapa Estratégico para la Atención al Paciente Externo", diseñaron  una estrategia para favorecer el desarrollo y expansión de la Telefarmacia en  España. Esta estrategia incluyó cuatro líneas de actuación. Para el desarrollo  de la primera, en marzo de 2021 se llevó a cabo una convocatoria dirigida a los socios de la Sociedad Española de Farmacia Hospitalaria con objeto de  desarrollar siete documentos de apoyo metodológico destinados a cubrir las  necesidades para la implantación de la Telefarmacia, entre las cuales se  consideraron como claves el desarrollo de una guía específica para  profesionales y otra para pacientes. Las guías se desarrollaron en cuatro fases  entre mayo y octubre de 2021: revisión de literatura, entrevistas de consenso,  desarrollo de improtalleres de trabajo online y debate y, por último,  laboración y validación de los documentos finales. Una vez elaborado el borrador  definitivo se llevó a cabo una valoración pública de sugerencias y alegaciones durante un mes, así como una presentación al comité de pacientes de la Sociedad Española de Farmacia Hospitalaria. RESULTADOS: La guía para profesionales se ha orientado para dar las pautas  para el desarrollo e implementación de programas de Telefarmacia en cada uno de sus ámbitos de aplicación. Está estructurada de forma que resalte los  objetivos concretos, los beneficios asistenciales, las herramientas necesarias,  así como las claves para implantarla. La guía para pacientes se ha desarrollado como herramienta complementaria a la atención farmacéutica presencial desde la visión del paciente, destacando las herramientas más comúnmente  utilizadas, los potenciales beneficios y las claves para facilitar la comprensión  sobre la finalidad y uso de estas intervenciones. CONCLUSIONES: Se ha desarrollado una guía práctica de apoyo, tanto a profesionales como a pacientes, para estandarizar el desarrollo, implantación  y expansión de la Telefarmacia en todos sus ámbitos de  actuación. La guía pretende ayudar al colectivo de farmacia hospitalaria a  alcanzar los potenciales beneficios de una herramienta que se presenta como  complementaria a la atención farmacéutica presencial.

Rapid Differentiation of Unfrozen and Frozen-Thawed Tuna with Non-Destructive Methods and Classification Models: Bioelectrical Impedance Analysis (BIA), Near-Infrared Spectroscopy (NIR) and Time Domain Reflectometry (TDR).

The performances of three non-destructive sensors, based on different principles, bioelectrical impedance analysis (BIA), near-infrared spectroscopy (NIR) and time domain reflectometry (TDR), were studied to discriminate between unfrozen and frozen-thawed fish. Bigeye tuna (Thunnus obesus) was selected as a model to evaluate these technologies. The addition of water and additives is usual in the fish industry, thus, in order to have a wide range of possible commercial conditions, some samples were injected with different water solutions (based on different concentrations of salt, polyphosphates and a protein hydrolysate solution). Three different models, based on partial least squares discriminant analysis (PLS-DA), were developed for each technology. This is a linear classification method that combines the properties of partial least squares (PLS) regression with the classification power of a discriminant technique. The results obtained in the evaluation of the test set were satisfactory for all the sensors, giving NIR the best performance (accuracy = 0.91, error rate = 0.10). Nevertheless, the classification accomplished with BIA and TDR data resulted also satisfactory and almost equally as good, with accuracies of 0.88 and 0.86 and error rates of 0.14 and 0.15, respectively. This work opens new possibilities to discriminate between unfrozen and frozen-thawed fish samples with different non-destructive alternatives, regardless of whether or not they have added water.

A New Rapid Method for the Authentication of Common Octopus (Octopus vulgaris) in Seafood Products Using Recombinase Polymerase Amplification (RPA) and Lateral Flow Assay (LFA).

The common octopus (Octopus vulgaris) is a highly valued cephalopod species which is marketed with different grades of processing, such as frozen, cooked or even canned, and is likely to be mislabeled. Some molecular methods have been developed for the authentication of these products, but they are either labor-intensive and/or require specialized equipment and personnel. This work describes a newly designed rapid, sensitive and easy-to-use method for the detection of Octopus vulgaris in food products, based on Recombinase Polymerase Amplification (RPA) and a detection using a Lateral Flow assay (LFA). After studying several gene markers, a system of primers and nfo-probe was designed in the COI (Cytochrome Oxidase I) region and was successfully tested in 32 reference samples (covering 14 species) and 32 commercial products, after optimization. The method was also validated in a ring trial with eight European laboratories and represents a useful tool for food authenticity control at all levels of the value chain.

A Real-Time PCR Method for the Authentication of Common Cuttlefish (Sepia officinalis) in Food Products.

Cephalopods are very relevant food resources. The common cuttlefish (Sepia officinalis) is highly appreciated by consumers and there is a lack of rapid methods for its authentication in food products. We introduce a new minor groove binding (MGB) TaqMan real-time PCR (Polymerase Chain Reaction) method for the authentication of S. officinalis in food products to amplify a 122 base pairs (bp) fragment of the mitochondrial COI (Cytochrome Oxidase I) region. Reference and commercial samples of S. officinalis showed a threshold cycle (Ct) mean of 14.40, while the rest of the species examined did not amplify, or showed a significantly different Ct (p < 0.001). The calculated efficiency of the system was 101%, and the minimum DNA quantity detected was 10-4 ng. No cross-reactivity was detected with any other species, thus, the designed method differentiates S. officinalis from other species of the genus Sepia and other cephalopod species and works for fresh, frozen, grilled, cooked and canned samples of Sepia spp. The method has proved to be reliable and rapid, and it may prove to be a useful tool for the control of fraud in cuttlefish products.

Adherence to treatment with adalimumab, golimumab and ustekinumab in patients with inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel disease comprises a group of chronic relapsing inflammatory disorders affecting the bowel. In the last  decade, the advent of biological drugs brought about a drastic change in the treatment of the disease. Adalimumab, golimumab and ustekinumab are three biologic agents that patients can self-administer  subcutaneously after collecting them from the pharmacy department.  However, for the treatment to be effective, adherence is paramount.  The purpose of the present study is to evaluate adherence in patients  who collected all three drugs from the dispensary of a tertiary care  hospital. METHOD: A cross-sectional observational analysis was carried out of patients who had been receiving treatment with adalimumab,  golimumab and ustekinumab for at least four months. The medication  possession ratio was calculated based on information extracted from the  pharmacy dispensing records. Patients with a ratio ≤ 85% were  enrolled in the study and asked to respond to Morisky-Green Medication  Adherence Questionnaire. Results: One-hundred and seventy-eight patients were included, of whom 60.1% (107) were male and 30.9% (55) had been treated  previously with other biologics. According to the pharmacy dispensing  records, mean adherence was 91.79%, with 45 patients (25.28%)  classified as scarcely compliant (≤ 85%). The Morisky-Green Medication  Adherence Questionnaire revealed that carelessness about administering the drug at the right time and forgetfulness were the main reasons for  therapeutic non-adherence. Female sex (odds ratio 0.42; p = 0.013)  and lengthy treatments (p = 0.002) were associated to lower adherence rates. CONCLUSIONS: Although most patients in the studied population were seen to be compliant, low levels of adherence were observed in a  number of patients who would benefit from interventions aimed at  boosting their adherence. It must be said, however, that the statistical  power of this study should be enhanced in order to increase the  significance of the results obtained.

Objetivo: La enfermedad inflamatoria intestinal es un grupo de  rastornos crónicos, inflamatorios y recidivantes que afectan al intestino.  En la última década, los fármacos biológicos han supuesto un gran  cambio en la terapia de esta enfermedad. Adalimumab, golimumab y  ustekinumab son tres de ellos que se administran por vía subcutánea  tras su dispensación en los servicios de farmacia de los hospitales. Para  que se alcance la efectividad del tratamiento es necesaria una adecuada adherencia al mismo. El objetivo del presente trabajo fue evaluar la  adherencia en pacientes que recogían los tres fármacos en el servicio de farmacia de un hospital de tercer nivel.Método: Se realizó un estudio analítico observacional de corte  transversal en el que se incluyó a pacientes que recibían tratamiento  con los anteriores fármacos durante al menos cuatro meses. Se recogió  la tasa de posesión de la medicación proporcionada por el registro de  dispensaciones y se seleccionó a los pacientes que presentaron un valor  inferior o igual al 85%. A estos pacientes se les aplicó el cuestionario de  medida del cumplimiento terapéutico de Morisky-Green.Resultados: Se incluyeron 178 pacientes, de los cuales el 60,1% (107)  fueron hombres y el 30,9% (55) habían sido tratados con otros  fármacos biológicos previamente. La adherencia media, según el  registro de dispensaciones, fue del 91,79% y se clasificó a 45 pacientes  (25,28%) como mal adherentes (≤ 85%). La no administración en la  fecha indicada y el olvido se identificaron como principales razones de la falta de cumplimiento terapéutico según el resultado del test de  Morisky-Green. El sexo femenino (odds ratio 0,42; p = 0,013) y la  duración del tratamiento (p = 0,002) se asociaron a una peor  adherencia a la medicación.Conclusiones: El porcentaje de adherencia obtenido resultó elevado en la población de estudio, pero se identificaron pacientes mal cumplidores susceptibles de recibir intervenciones para mejorar su  adherencia. No obstante, se debería aumentar la potencia estadística  para mejorar la validez de los resultados obtenidos.

Tuna labels matter in Europe: Mislabelling rates in different tuna products.

Tuna fisheries and processing represent economic activities of paramount importance around the world. Most of these products are traded for human consumption and in general are highly demanded commodities. However, not all tuna products achieve the same market price, some consumers are willing to pay a huge amount of money for certain species (i.e. Japanese market for Bluefin tuna) while other species are rather affordable (i.e. Skipjack tuna), therefore mislabelling has been observed frequently. We collected and analysed 545 tuna samples in six European countries, including fresh, frozen and canned products, and we have investigated whether or not these products were correctly labelled under European and national legislations. We found an overall mislabelling rate of 6.79%; in particular, 6.70% of the fresh and frozen tuna products and 7.84% of canned tuna were mislabelled, and only in the case of fresh and frozen tuna samples significant differences among countries were found. Mislabelling rates for Atlantic Bluefin tuna labelled products were very high, ranging from 50 up to 100%. In general, mislabelling was higher when specific names were included in the labels. The "tuna" umbrella term is a very popular one with consumers, but also one that remains vulnerable to ambiguity, hampering efforts towards market transparency and with potential negative consequences to the adequate management of tuna species stocks.

Assessment of the labelling accuracy of spanish semipreserved anchovies products by FINS (forensically informative nucleotide sequencing).

Anchovies have been traditionally captured and processed for human consumption for millennia. In the case of Spain, ripened and salted anchovies are a delicacy, which, in some cases, can reach high commercial values. Although there have been a number of studies presenting DNA methodologies for the identification of anchovies, this is one of the first studies investigating the level of mislabelling in this kind of products in Europe. Sixty-three commercial semipreserved anchovy products were collected in different types of food markets in four Spanish cities to check labelling accuracy. Species determination in these commercial products was performed by sequencing two different cyt-b mitochondrial DNA fragments. Results revealed mislabelling levels higher than 15%, what authors consider relatively high considering the importance of the product. The most frequent substitute species was the Argentine anchovy, Engraulis anchoita, which can be interpreted as an economic fraud.

Development of a multiplex PCR-ELISA method for the genetic authentication of Thunnus species and Katsuwonus pelamis in food products.

In the present work a PCR-ELISA technique for the authentication of Thunnus species was developed. This method is composed by four systems that can be used in a hierarchical way allowing the identification of several scombroids species; or each individual system independently. The hierarchical strategy, proposes a first step, to assign one sample to the Thunnus genus. Next, if the result is positive, several tests can be applied to assign the sample to some particular species of the Thunnus genus. In the case that the result is negative (absence of Thunnus species), it is possible to verify if Katsuwonus pelamis is included in the sample. The method even allows the detection of mixtures of these species in relatively low amounts (up to 1%). Finally, this method was applied to 11 commercial samples to verify the labelling status of tuna products in the market, detecting that 18% were mislabelling.

Identification of Atlantic cod (Gadus morhua), ling (Molva molva), and Alaska pollock (Gadus chalcogrammus) by PCR-ELISA using duplex PCR.

Species-specific PCR-ELISA assays for the identification of Atlantic cod (Gadus morhua), Alaska pollock (Gadus chalcogrammus), and ling (Molva molva) in food products have been developed. The method, comprising a set of primers common to the first two species, a set of primers for M. molva, and a probe for each species, was designed using ND4 and cytochrome b genes as molecular markers. The sensitivity and selectivity were then determined for each assay. These assays were afterward used to analyze DNA extracted from commercial fish products. The presence of the target species was successfully detected in all analyzed samples, demonstrating the applicability of this method to the analysis of food products.

Development of a real-time PCR method for the identification of Atlantic mackerel (Scomber scombrus).

A Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P<0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel products.