Barriers and Strategies to Optimize the Use of Glucagon-Like Peptide 1 Receptor Agonists in People with Type 2 Diabetes and High Cardiovascular Risk or Established Cardiovascular Disease: A Delphi Consensus in Spain.

INTRODUCTION: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are effective for glycemic control, with many also demonstrating cardiovascular (CV) benefit, in people with type 2 diabetes (T2D). This study aimed to find a consensus on the barriers and strategies for the optimal use of GLP-1 RAs in people with T2D and high CV risk or established cardiovascular disease (CVD) in Spain. METHODS: A two-round Delphi survey (53 questions) was conducted among members of four national scientific societies in Spain, including physicians experienced in the management of people with T2D. The degree of consensus was evaluated with a 7-point Likert scale, establishing consensus when ≥ 70% of the panelists agreed (6-7) or disagreed (1-2). RESULTS: A total of 97 physicians participated in the first round (endocrinology: 34%, family and community medicine: 21%, internal medicine: 23%, and cardiology: 23%), and 96 in the second round. The main barriers identified were: therapeutic inertia and late use of GLP-1 RAs; lack of a comprehensive approach to CV risk; lack of knowledge on the usefulness of GLP-1 RAs in CVD prevention and treatment; and economic/administrative barriers. Strategies with a highest consensus included: the need to establish simple protocols that integrate awareness of CV risk monitoring; training professionals and patients; and the use of new technologies. CONCLUSION: Physicians identified clinical, healthcare, and economic/administrative barriers that limit the use of GLP-1 RAs in people with T2D and high CV risk or established CVD in Spain, highlighting the importance of integrating these therapies according to clinical practice guidelines.

Epidemiological, Clinical, Patient-Reported and Economic Burden of Inflammatory Bowel Disease (Ulcerative colitis and Crohn's disease) in Spain: A Systematic Review.

INTRODUCTION: This study describes the epidemiological, clinical, patient-reported and economic burden of inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), in Spain. METHODS: A systematic review was performed of observational studies reporting the epidemiological, clinical, patient-reported and economic burden of IBD in the Spanish population, from 2011 to 2021. Original articles and conference abstracts published in English or Spanish were eligible. RESULTS: A total of 45 publications were included in the review. The incidence of IBD in adults ranged from 9.6 to 44.3 per 100,000 inhabitants (4.6 to 18.5 for CD and 3.4 to 26.5 for UC). The incidence increased between 1.5- and twofold from 2000 to 2016 (regionally). Up to 6.0% (CD) and 3.0% (UC) IBD-associated mortality was reported. Disease onset predominantly occurs between 30 and 40 years (more delayed for UC than CD). Most frequently reported gastrointestinal manifestations are rectal bleeding in UC and weight loss in CD. Extraintestinal manifestations (EIM) have been described in up to 47.4% of patients with CD and 48.1% of patients with UC. Psychiatric comorbidities were frequently reported in both CD and UC (depression up to 20% and anxiety up to 11%). Reduced health-related quality of life (HRQoL) compared to the general population was reported. Significant symptomatology was associated with high levels of anxiety, depression, stress and lower HRQoL. Main healthcare resources reported were emergency department visits (24.0%), hospitalization (14.7%), surgery (up to 11%) and use of biologics (up to 60%), especially in CD. Direct and indirect annual costs per patient with UC were €1754.1 and €399.3, respectively. CONCLUSION: Patients with CD and UC present a high disease burden which negatively impacts their HRQoL, leading to elevated use of resources.

[Opinions and experiences of healthcare professionals in the nutritional management of oncology patients: the ONA study]. / Opiniones y experiencias de profesionales sanitarios en el manejo nutricional del paciente oncológico: estudio ONA.

Introduction: Objectives: an inadequate approach to prevent malnutrition in cancer patients may worsen their quality of life and reduce their response to treatment. This study aims to describe the nutritional management of cancer patients in clinical practice, as well as the opinions of the healthcare professionals (HCPs) involved. Methods: this was an observational, descriptive, cross-sectional study addressed to HCPs in the Spanish healthcare setting. The online questionnaire was designed based on a literature review, one focus group of patients (n = 6), and the experience of the multidisciplinary scientific committee (n = 5), and was distributed by the scientific societies endorsing the study. Results: a total of 461 HCPs answered the survey. Most of them considered that a nutrition expert (95.0 %) is essential for the nutritional management of patients. However, 22.8 % of HCPs did not have access to this expert, and only 49.0 % had received training. Nutritional screening or patient referral for screening was performed by 58.4 % of HCPs. Of the total of HCPs, 86.6 % stated that nutritional information is provided to patients and considered them moderately satisfied with the information received. In malnourished patients or in those at risk of malnutrition, a complete nutritional assessment was performed by HCPs (73.1 %). Most HCPs (87.4 %) reported prescribing or recommending nutritional support if needed, and assessing adherence (86.8 %). Conclusions: despite malnutrition being a common problem in cancer patients, almost half of professionals do not perform any nutritional screening. In addition, patient information and assessment of nutritional adherence appear to be suboptimal.

Introducción: Objetivos: un abordaje inadecuado de la desnutrición en el paciente con cáncer puede conducir a un empeoramiento de su calidad de vida y una respuesta deficiente al tratamiento. El estudio ONA (Oncología, Nutrición y Adherencia) tiene como objetivo describir el manejo nutricional del paciente con cáncer en la práctica clínica, así como las opiniones de los profesionales sanitarios involucrados en el mismo. Métodos: estudio observacional, descriptivo y transversal dirigido a profesionales sanitarios españoles. El cuestionario online fue diseñado a partir de una revisión bibliográfica, un grupo focal de pacientes (n = 6) y un comité científico multidisciplinar (n = 5), y distribuido por las sociedades científicas que avalan el estudio. Resultados: de los 461 profesionales sanitarios participantes, el 95,0 % consideraron fundamental la figura del profesional sanitario con formación específica en nutrición, pero el 22,8 % no tenían acceso a ella y solo el 49,0 % habían recibido formación. El 58,4 % afirmaron realizar el cribado nutricional o derivar al paciente para este fin. El 86,6 % de los participantes indicaron que se informa al paciente sobre aspectos nutricionales y consideraron que este estaba moderadamente satisfecho con la información recibida. En caso de detectarse desnutrición o riesgo de desnutrición, los profesionales afirmaron realizar una evaluación nutricional completa (73,1 %) y, de necesitarse soporte nutricional, este se prescribiría/recomendaría (87,4 %), evaluándose la adherencia al mismo (86,8 %). Conclusiones: a pesar de que la desnutrición es un problema común en el paciente con cáncer, casi la mitad de los profesionales no realizan un cribado nutricional. Además, el proceso de información y evaluación de la adherencia nutricional es subóptimo.

Management of prediabetes from the perspective of Spanish physicians and community pharmacists: Detecta2 study.

INTRODUCTION: Prediabetes is a high-risk state for diabetes. The study aims to describe routine clinical practice and the views of physicians and pharmacists on prediabetes management. MATERIALS AND METHODS: An observational, descriptive, cross-sectional study was conducted using a structured questionnaire. RESULTS: A total of 410 physicians and 393 pharmacists completed the questionnaire. Self-adherence to clinical practice guidelines (CPGs) was reported by 51.5% and 23.2% of physicians and pharmacists, respectively. Less than 60% of participants defined prediabetes according to main CPG. Regarding the use of screening strategies to detect prediabetes (physicians: 96%; pharmacists: 42.1%), reports indicate the opportunistic strategy is widely employed (≥75%) whereas systematic screening is unusual (<20%). Changes in lifestyle were deemed essential by almost all participants (≥95%), but in clinical practice only 58.3% of healthcare centers and 28.0% of pharmacies were found to provide awareness-raising/instruction. The role of pharmacists in the prevention of prediabetes/diabetes was judged useful by most participants. CONCLUSIONS: Use of CPG, systematic prediabetes screening strategies, and specific strategies for patient education are scarce. The support of community pharmacists in prediabetes management was well valued. Therefore, it is crucial that the lines of action followed by both physicians and pharmacists align with each other and with the CPG.

Management of prediabetes from the perspective of Spanish physicians and community pharmacists: Detecta2 study.

INTRODUCTION: Prediabetes is a high-risk state for diabetes. The study aims to describe routine clinical practice and the views of physicians and pharmacists on prediabetes management. MATERIALS AND METHODS: An observational, descriptive, cross-sectional study was conducted using a structured questionnaire. RESULTS: A total of 410 physicians and 393 pharmacists completed the questionnaire. Self-adherence to clinical practice guidelines (CPGs) was reported by 51.5% and 23.2% of physicians and pharmacists, respectively. Less than 60% of participants defined prediabetes according to main CPG. Regarding the use of screening strategies to detect prediabetes (physicians: 96%; pharmacists: 42.1%), reports indicate the opportunistic strategy is widely employed (≥75%) whereas systematic screening is unusual (<20%). Changes in lifestyle were deemed essential by almost all participants (≥95%), but in clinical practice only 58.3% of healthcare centers and 28.0% of pharmacies were found to provide awareness-raising/instruction. The role of pharmacists in the prevention of prediabetes/diabetes was judged useful by most participants. CONCLUSIONS: Use of CPG, systematic prediabetes screening strategies, and specific strategies for patient education are scarce. The support of community pharmacists in prediabetes management was well valued. Therefore, it is crucial that the lines of action followed by both physicians and pharmacists align with each other and with the CPG.

Cost, microbiological, and nutritional properties of pureed food production in nursing homes. The ABADIA Study.

INTRODUCTION: Introduction: although nutritional differences between different types of texture-modified diet (TMD) have been evaluated, the resources and costs associated with their preparation have been less studied. Objective: to describe the nutritional, microbiological properties and costs of: 1) in-home produced pureed food (hTMD); 2) concentrated nutrient-dense commercial food products, hand-blended (cTMD); 3) food prepared using the MixxPro® automatic food mixer (cTMD-Mix). Methods: an observational, prospective study carried out in three geriatric nursing-homes. Patients ≥ 65 years, receiving TMD, with a stable clinical condition, estimated survival/expected internment > 1 month, and sufficient cognitive capacity were included. The following data were recorded: 1) patient socio-demographic and clinical variables; 2) TMD compliance and symptoms related to dysphagia during the meal; 3) patient appetite; and 4) kitchen information and resources used to prepare a TMD. Results: sixty-two residents were included (65.0 % women, 88.3 years (SD: 9.3); 43.5 % malnourished, 79.0 % with good appetite). The proportion of food eaten/median kcal served/portion/mean kcal consumed were: hTMD: 95.5 % (SD: 10.7)/92.4 kcal (IQR: 75.6-128.1)/88.2 kcal (IQR: 72.2-122.3); cTMD: 89.2 % (SD: 15.9)/323.4 kcal (IQR: 284.2-454.1)/288.5 kcal (IQR: 253.5-325.1); and cTMD-Mix: 80.3 % (SD: 21.4)/358.0 kcal (IQR: 344.0-372.1)/287.5 kcal (IQR: 276.5-298.8). No microorganisms were detected. The average time spent in preparing each portion and its costs were: hTMD: 11.2 min (SD: 3.89)/€2.33 (SD: 0.63); cTMD: 1.7 min (SD: 0.28)/€2.01 (SD: 0.39); and cTMD-Mix: 1.6 min (SD: 0.00)/€2.00 (SD: 0.33). Conclusions: in patients with dysphagia and/or chewing difficulties, concentrated nutrient-dense food products, particularly those produced using the MixxPro® automatic food mixer, ensure a high caloric intake and allow quick and safe food preparation.

INTRODUCCIÓN: Introducción: aunque existe evidencia acerca de las diferencias nutricionales entre los distintos tipos de dieta de textura modificada (DTM), los recursos y los costos asociados a su preparación se han estudiado menos. Objetivo: describir las propiedades nutricionales, las microbiológicas y los costes de: 1) una dieta triturada de manera artesanal (hDTM); 2) una dieta preparada con alimentación básica adaptada (ABA) (cDTM); y 3) una ABA preparada con el mezclador automático de alimentos MixxPro® (cDTM-Mix). Métodos: estudio observacional prospectivo realizado en tres residencias. Se incluyeron pacientes ≥ de 65 años que recibían DTM, con estado clínico estable, con supervivencia/internamiento estimado > 1 mes y capacidad cognitiva suficiente. Se registraron: 1) las variables sociodemográficas y clínicas del paciente; 2) el cumplimiento y los síntomas relacionados con la disfagia durante la comida; 3) el apetito del paciente, y 4) la información de la cocina y los recursos utilizados para preparar la DTM. Resultados: se incluyeron 62 residentes (65,0 % mujeres, 88,3 años (SD: 9,3), 43,5 % desnutridos, 79,0 % con buen apetito). La proporción de alimentos consumidos/mediana de kcal servidas/porción/media de kcal media consumidas fueron: hDTM 95,5 % (SD: 10,7)/92,4 kcal (IQR: 75,6-128,1)/88,2 kcal (IQR: 72,2-122,3); cDTM: 89.2 % (SD: 15.9)/323,4 kcal (IQR: 284.2-454.1)/288,5 kcal (IQR: 253.5-325.1), y cDTM-Mix: 80,3 % (SD: 21.4)/358,0 kcal (IQR: 344.0-372.1)/287,5 kcal (IQR: 276.5-298.8). No se detectaron microorganismos. El tiempo medio empleado en la preparación y el coste por porción fueron: hDTM: 11,2 min (SD: 3,89)/2,33 € (SD: 0,63); cDTM: 1,7 min (SD: 0,28)/2,01 € (SD: 0,39), y cDTM-Mix: 1,6 min (SD: 0,00)/2,00 € (SD: 0,33). Conclusiones: en los pacientes con disfagia y/o dificultades para masticar, los productos de ABA comerciales, en particular los que se producen con el mezclador automático de alimentos MixxPro®, aseguran una elevada ingesta calórica y permiten una preparación rápida y segura.

Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease.

Current molecular PCR-based techniques used for detecting Streptococcus pneumoniae, the causative pathogen of invasive pneumococcal disease (IPD), are accurate but have a run time of several hours. We aimed to develop and validate a novel real-time loop mediated amplification (LAMP) assay for rapid detection of pneumococcus in normally sterile samples with accuracy comparable to a gold standard real-time PCR. Conserved regions of lytA were used for the design of the LAMP test. Analytical validation included assessment of linearity, limit of detection (LOD), intra-assay and inter-assay precision and analytical specificity, which was evaluated by using reference strain S. pneumoniae R6 and a quality control panel. Clinical performance was assessed on all samples collected from children with suspicion of IPD attended in Hospital Sant Joan de Deu (Barcelona, Spain) during the period April-September 2015. Fresh samples were analyzed after DNA extraction. The following values of analytical parameters were determined: linearity within the range 108-104 copies/mL; limit of detection, 5·103 copies/mL; intra- and inter-assay precision measured by mean coefficient of variance, 3.61 and 6.59%; analytical specificity, 9/9 pathogens similar to S. pneumoniae and 14/14 strains of different S. pneumoniae serotypes correctly identified as negative and positive results, respectively. Diagnostic sensitivity and specificity values were 100.0 and 99.3%. Median time of DNA amplification was 15 min. The new LAMP assay showed to have similar accuracy as PCR while being 5-fold faster and could become a useful diagnostic tool for early diagnosis of IPD.

Cross-sectional study about the use of telemedicine for type 2 diabetes mellitus management in Spain: patient's perspective. The EnREDa2 Study.

OBJECTIVES: The usefulness of telemedicine (TM) in type 2 diabetes mellitus (T2DM) has been discussed in recent years. The aim of this study is to describe patients' perceptions about TM and to identify preferences on TM resources, in Spain. DESIGN: An observational, cross-sectional study was conducted using a structured questionnaire. PARTICIPANTS: 1036 patients with T2DM accepted to participate in the study (response rate: 68%). RESULTS: Blood glucose values were recorded by 85.9% of the patients while data such as lifestyle habits were only recorded by 14.4% of the patients. Previous experience in TM was reported by 9.8% of the patients, out of which 70.5% were satisfied with its service and 73.5% considered that the use of TM had optimised their T2DM management. However, most of these patients noted aspects to be improved such as user-friendliness (81.4%), interaction with the medical team (78.4%) and time required for recording/transferring data (78.4%). Experienced patients had better perception about TM usefulness than naïve patients for all listed aspects (p<0.05). Among naïve patients, 38.2% expressed their willingness to participate in TM programmes, but only 4.7% were invited to do so. Patients considered that physicians' (77.5%) and pharmacists' (75.5%) encouragement can boost the use of TM. CONCLUSIONS: In Spain, nearly 10% of patients with T2DM have experience with TM and it is well accepted, especially one based on glucometers. Nevertheless, in order to promote TM use, easier and time-saving programmes for patient-physician interaction should be optimised.

Physicians' and Pharmacists' Clinical Considerations for Elderly Patients with Type 2 Diabetes Mellitus: The IMPLICA2 Study.

BACKGROUND: Management of elderly patients with type 2 diabetes mellitus (T2DM) is complex due to their age-related conditions. Several clinical guidelines provide specific recommendations for management of these patients but little is known about their implementation in clinical practice. OBJECTIVE: To describe physician and community pharmacist perceptions and routine clinical practice in the management of elderly T2DM patients. METHODS: Cross-sectional study. RESULTS: A total of 993 physicians and 999 community pharmacists completed the questionnaire. More physicians than pharmacists agreed on the need to establish more flexible HbA1c targets for elderly (79.4% vs. 30.6%; p < 0.001) and frail (92.6% vs. 31.4%; p < 0.001) patients than for the general diabetic population. HbA1c targets < 7.5% for elderly patients and < 8.5% for frail patients (as recommended by the principle guidelines) were set by 38.9% and 28.7% of physicians, respectively. Furthermore, 62.8% of physicians stated they follow guideline recommendations but, based on their prescription decisions for hypothetical patients, less than 50% were aligned with them. In addition, 73.1% of physicians monitor treatment adherence, mainly by using dispensing control (59.1%). Specific nutritional approaches for elderly patients are provided by 62.9% of physicians and 56.0% of pharmacists, whilst 57.4% and 21.7%, respectively, deliver specific physical exercise programs. CONCLUSIONS: Low adherence to guideline recommendations (i.e. setting more stringent HbA1c targets or delaying treatment intensification) may lead to suboptimal glycaemic control in elderly patients. The standardization of processes, extensive monitoring of patient treatment adherence and providing advice regarding specific personal lifestyle habits may improve the management of elderly T2DM patients.

Defining a set of standardised outcome measures for newly diagnosed patients with multiple myeloma using the Delphi consensus method: the IMPORTA project.

OBJECTIVE: To define a standard set of outcomes and the most appropriate instruments to measure them for managing newly diagnosed patients with multiple myeloma (MM). METHODS: A literature review and five discussion groups facilitated the design of two-round Delphi questionnaire. Delphi panellists (haematologists, hospital pharmacists and patients) were identified by the scientific committee, the Spanish Program of Haematology Treatments Foundation, the Spanish Society of Hospital Pharmacies and the Spanish Community of Patients with MM. Panellist's perception about outcomes' suitability and feasibility of use was assessed on a seven-point Likert scale. Consensus was reached when at least 75% of the respondents reached agreement or disagreement. A scientific committee led the project. RESULTS: Fifty-one and 45 panellists participated in the first and second Delphi rounds, respectively. Consensus was reached to use overall survival, progression-free survival, minimal residual disease and treatment response to assess survival and disease control. Panellists agreed to measure health-related quality of life, pain, performance status, fatigue, psychosocial status, symptoms, self-perception on body image, sexuality and preferences/satisfaction. However, panellist did not reach consensus about the feasibility of assessing in routine practice psychosocial status, symptoms, self-perception on body image and sexuality. Consensus was reached to collect patient-reported outcomes through the European Organisation for the Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ) Core questionnaire 30 (C30), three items from EORTC-QLQ-Multiple Myeloma (MY20) and EORTC-QLQ-Breast Cancer (BR23), pain Visual Analogue Scale, Morisky-Green and ad hoc questions about patients' preferences/satisfaction. CONCLUSIONS: A consensual standard set of outcomes for managing newly diagnosed patients with MM has been defined. The feasibility of its implementation in routine practice will be assessed in a future pilot study.

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB.

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening-closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.

Differences in Bordetella pertussis DNA load according to clinical and epidemiological characteristics of patients with whooping cough.

OBJECTIVE: To identify associations between nasopharyngeal Bordetella pertussis DNA load and clinical and epidemiological characteristics and evaluate DNA load prognostic value in pertussis severity. METHODS: Prospective observational multi-centre study including nasopharyngeal samples positive to pertussis DNA by real-time PCR collected from children and adult patients in more than 200 health centres of Catalonia (Spain) during 2012-2013. RESULTS: B. pertussis load was inversely correlated with age (rho = -0.32, p < 0.001), time to diagnosis (rho = -0.33, p < 0.001) and number of symptoms (rho = 0.13, p = 0.002). Median bacterial load was significantly higher in inpatients versus outpatients (4.91 vs. 2.55 log10 CFU/mL, p < 0.001), patients with complications versus those without (6.05 vs. 2.82 log10 CFU/mL, p < 0.001), disease incidence in summer and autumn versus spring and winter (3.50 vs. 2.21 log10 CFU/mL, p = 0.002), and unvaccinated-partially vaccinated patients versus vaccinated (4.20 vs. 2.76 log10 CFU/mL, p = 0.004). A logistic regression model including bacterial load and other candidate prognostic factors showed good prediction for hospital care (AUC = 0.94) although only age and unvaccinated status were found to be prognostic factors. CONCLUSIONS: We observed strong positive associations of nasopharyngeal bacterial load with severity outcomes of hospitalisation and occurrence of complications. Bacterial load and other independent variables contributed to an accurate prognostic model for hospitalisation.

Validation of a loop-mediated isothermal amplification assay for rapid diagnosis of pertussis infection in nasopharyngeal samples.

OBJECTIVE: To develop and validate a novel loop-mediated amplification (LAMP) assay for rapid diagnosis (<1 hour) of whooping cough in nasopharyngeal samples versus the gold standard: real-time PCR. METHODS: The study included all nasopharyngeal samples (n = 213) collected from children with clinical suspicion of pertussis admitted to Children's University Hospital Sant Joan de Déu (Barcelona, Spain) during July-December 2014. Fresh samples were routinely analyzed by real-time PCR and stored for retrospective LAMP analysis, following an easy 30 minute DNA extraction step by Chelex-100. RESULTS: Performance results of the LAMP assay were: linearity, 10(5)-10(1) CFU/ml; Limit of Detection, 2 CFU/ml; precision (mean CV), 7.38%; diagnostic sensitivity, 96.55%; diagnostic specificity, 99.46%; time to detection, 12-30 minutes. CONCLUSION: The new test was shown to be 2.5-fold faster than real-time PCR while maintaining similar levels of analytical and clinical performance. Therefore it could become a useful diagnostic tool for molecular point-of-care testing.

A novel chimeric phage lysin with high in vitro and in vivo bactericidal activity against Streptococcus pneumoniae.

OBJECTIVES: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. METHODS: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 µg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. RESULTS: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 µg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 µg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. CONCLUSIONS: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

Recurrent invasive pneumococcal disease in children: underlying clinical conditions, and immunological and microbiological characteristics.

PURPOSE: Clinical, immunological and microbiological characteristics of recurrent invasive pneumococcal disease (IPD) in children were evaluated, differentiating relapse from reinfection, in order to identify specific risk factors for both conditions. METHODS: All patients <18 years-old with recurrent IPD admitted to a tertiary-care pediatric center from January 2004 to December 2011 were evaluated. An episode of IPD was defined as the presence of clinical findings of infection together with isolation and/or pneumococcal DNA detection by Real-Time PCR in any sterile body fluid. Recurrent IPD was defined as 2 or more episodes in the same individual at least 1 month apart. Among recurrent IPD, we differentiated relapse (same pneumococcal isolate) from reinfection. RESULTS: 593 patients were diagnosed with IPD and 10 patients died. Among survivors, 23 episodes of recurrent IPD were identified in 10 patients (1.7%). Meningitis was the most frequent form of recurrent IPD (10 episodes/4 children) followed by recurrent empyema (8 episodes/4 children). Three patients with recurrent empyema caused by the same pneumococcal clone ST306 were considered relapses and showed high bacterial load in their first episode. In contrast, all other episodes of recurrent IPD were considered reinfections. Overall, the rate of relapse of IPD was 0.5% and the rate of reinfection 1.2%. Five out of 7 patients with reinfection had an underlying risk factor: cerebrospinal fluid leak (n = 3), chemotherapy treatment (n = 1) and a homozygous mutation in MyD88 gene (n = 1). No predisposing risk factors were found in the remainder. CONCLUSIONS: recurrent IPD in children is a rare condition associated with an identifiable risk factor in case of reinfection in almost 80% of cases. In contrast, recurrent IPD with pleuropneumonia is usually a relapse of infection.

Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics.

Nucleic acid amplification techniques such as PCR have facilitated rapid and accurate diagnosis in central laboratories over the past years. PCR-based amplifications require high-precision instruments to perform thermal cycling reactions. Such equipment is bulky, expensive and complex to operate. Progressive advances in isothermal amplification chemistries, microfluidics and detectors miniaturisation are paving the way for the introduction and use of compact 'sample in-results out' diagnostic devices. However, this paradigm shift towards decentralised testing poses diverse technological, economic and organizational challenges both in industrialized and developing countries. This review describes the landscape of molecular isothermal diagnostic techniques for infectious diseases, their characteristics, current state of development, and available products, with a focus on new directions towards point-of-care applications.

Improving the lethal effect of cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module.

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from -14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-µg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.

Microarray studies reveal a 'differential response' to moderate or severe heat shock of the HrcA- and HspR-dependent systems in Corynebacterium glutamicum.

This article has been retracted at the request of Microbiology because identical bands for the 16S rRNA probe controls in the Northern blots were reported to correspond to experiments using different strains and experimental conditions in articles published in this journal and in Journal of Bacteriology over a period of 5 years.

Structural independence of conjugative coupling protein TrwB from its Type IV secretion machinery.

The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported.