Intra- and Extra-Hospital Dissemination of IMP-22-Producing Klebsiella pneumoniae in Northern Portugal: The Breach of the Hospital Frontier Toward the Community.

The emergence of infections (and colonization) with Enterobacteriaceae-producing carbapenemases is a threatening public health problem. In the last decades, we watched an isolated case becoming a brutal outbreak, a sporadic description becoming an endemic problem. The present study aims to highlight the dissemination of IMP-22-producing Klebsiella pneumoniae in the North of Portugal, through the phenotypic and genotypic characterization of isolates collected from hospitalized patients (n=5) and out-patients of the emergency ward of the same acute care hospital (n=2), and isolates responsible for the intestinal colonization of residents in a Long-Term Care Facility (n=4). Pulsed-field gel electrophoresis (PFGE) results, associated with conjugation experiments pointed to a pattern of both vertical and horizontal dissemination. Overall, and complementing other studies that give relevance to IMP-22-producing K. pneumoniae in the clinical settings, here we show for the first time the public health threatening breach of the hospital frontier of this resistance threat, toward the community.

OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution.

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide-sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

Sampling Methods and Risk Stratification Regarding Environmental Contamination by SARS-CoV-2.

INTRODUCTION: Transmission of COVID-19 through close contact and droplets is well established, but the influence of aerosol and surface contamination remains to be determined. Literature is scarce and inconsistent about the viable virus particles free-distance from infected patients, as well as about different swabbing methods for surface contamination evaluation. The aim of this study was to evaluate the most sensitive method for the assessment of surface contamination, classify the likelihood of environmental contamination in risk zones and compare the environmental contamination between oxygenation and ventilatory support. MATERIAL AND METHODS: Swabs from potentially contaminated surfaces in a COVID-19 ward, with patients treated with different types of oxygen and ventilatory support, were collected. Three types of swabs were compared in order to evaluate the most sensitive collection method. For risk zone categorization, areas were divided according to the distance from the patient. RESULTS: Of the 63 swabs collected, 17 (27%) tested positive for the presence of SARS-CoV-2. The highest positivity rate was observed with the sterile premoistened swab with saline (n = 8; 38%), but without statistically significant differences. The highest number of positive samples were collected from the high-risk zones, specifically those located one meter from the patient (n = 13; 48%), with statistically significant differences. Only the rooms of patients supported with non-invasive ventilation or high-flow nasal cannula had evidence of bedroom contamination, with 45% and 27% of swab positivity, with statistically significant differences. DISCUSSION: Our findings favour the premoistened swab without transport medium for surface contamination assessment, even though without statistical differences. A statistically significant trend supporting the division in risk zones, according to the distance from the patient, was also identified. The higher positivity rate from the non-invasive ventilation and high-flow nasal cannula bedrooms suggests a significant association between ventilatory strategies and surface contamination, probably due to higher particle dispersion. CONCLUSION: Our findings support the use of the sterile premoistened swab without preservation medium, the classification of risk areas considering the distance from the patient, and the variability of RNA dispersion between oxygenation and ventilatory support.

Introdução: A transmissão da COVID-19 através do contacto e gotículas está bem estabelecida, mas a importância da sua transmissão através do aerossol e da contaminação das superfícies permanece por determinar. A literatura é escassa e inconsistente em relação à distância mínima livre de partículas víricas, desde um paciente, e também acerca dos mais adequados métodos de colheita de zaragatoas para avaliação da contaminação das superfícies. Os objectivos deste estudo foram avaliar qual o método mais sensível para avaliação da contaminação de superfícies, classificar a contaminação ambiental de acordo com zonas de risco e comparar a contaminação ambiental sob diferentes dispositivos para oxigenoterapia e suporte ventilatório. Material e Métodos: Realizamos colheitas de zaragatoas em superfícies potencialmente contaminadas numa ala COVID-19, onde se encontravam doentes sob diferentes dispositivos para oxigenoterapia e suporte ventilatório. Para avaliar o método de recolha mais sensível para verificação da contaminação das superfícies, comparámos três tipos de zaragatoas. Para a classificação das zonas de risco, dividimos as áreas de acordo com a distância ao doente. Resultados: Das 63 zaragatoas, 17 (27%) testaram positivo para SARS-CoV-2 (27%). A maior positividade foi observada na zaragatoa estéril pré-humedecida com soro fisiológico (n = 8; 38%), mas sem significância estatística. O maior número de amostras positivas obteve-se nas zonas de alto risco, especialmente aquelas a um metro do paciente (n = 13; 48%), com diferenças significativas. Apenas os quartos dos doentes sob ventilação não invasiva e cânula nasal de alto fluxo tiveram evidência de contaminação com 45% e 27% de positividade das zaragatoas, e significância estatística. Discussão: Os nossos resultados favorecem a zaragatoa estéril pré-humedecida sem meio de preservação para avaliação da contaminação das superfícies, embora sem significância estatística. Os resultados suportam também com significância estatística a divisão em zonas de risco de acordo com a distância ao doente. A maior positividade obtida nos quartos dos pacientes que se encontravam a utilizar ventilação não invasiva e cânula nasal de alto fluxo sugere uma associação, com significância, entre as estratégias ventilatória e a contaminação ambiental, provavelmente relacionada com uma maior dispersão das partículas. Conclusão: Os nossos resultados apoiam o uso da zaragatoa estéril pré-humedecida sem meio de preservação, a classificação das áreas de risco considerando a distância ao doente, e a variabilidade da dispersão do RNA entre diferentes dispositivos para oxigenoterapia e ventilação.

Acute Alithiasic Cholecystitis and Human Herpes Virus Type-6 Infection: First Case.

A three-year-old male child presented with erythematous maculopapular nonpruritic generalized rash, poor feeding, vomiting, and cramping generalized abdominal pain. He was previously healthy and there was no family history of immunologic or other diseases. On examination he was afebrile, hemodynamically stable, with painful palpation of the right upper quadrant and positive Murphy's sign. Laboratory tests revealed elevated inflammatory markers, elevated aminotransferase activity, and features of cholestasis. Abdominal ultrasound showed gallbladder wall thickening of 8 mm with a positive sonographic Murphy's sign, without gallstones or pericholecystic fluid. Acute Alithiasic Cholecystitis (AAC) was diagnosed. Tests for underlying infectious causes were negative except positive blood specimen for Human Herpes Virus Type-6 (HHV-6) by polymerase chain reaction. With supportive therapy the child became progressively less symptomatic with gradual improvement. The child was discharged on the sixth day, asymptomatic and with improved analytic values. Two months later he had IgM negative and IgG positive antibodies (1/160) for HHV-6, which confirmed the diagnosis of previous infection. In a six-month follow-up period he remains asymptomatic. To the best of our knowledge, this represents the first case of AAC associated with HHV-6 infection.

Analysis of a local HIV-1 epidemic in portugal highlights established transmission of non-B and non-G subtypes.

The existing data support Portugal as the western European country with the highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1-infected individuals followed at the reference hospital of the province of Minho, Portugal, at which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%) and the C (14.5%), F1 (7.3%), and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with the A1 and F1 subtypes increased over the years compared with those with B, G, or C subtypes or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of the B and G subtypes were found. However, transmission clusters of non-B and non-G viruses among native individuals were also found, with the dates of the most recent common ancestor estimated to be in the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and are mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

High level biosynthesis of a silk-elastin-like protein in E. coli.

Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications, yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L(-1) with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high preinduction growth rates and low inducer and oxygen concentrations are allowed whereas reduced postinduction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible, and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace.

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation.

BACKGROUND: Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system. RESULTS: This study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6-7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained. CONCLUSIONS: We have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded ß-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.

G2P[4] the most prevalent rotavirus genotype in 2007 winter season in an European non-vaccinated population.

BACKGROUND: Recently, a high prevalence of G2P[4] rotavirus (RV) infection was reported from Brazil, and linked with the universal RV vaccination programme that used the G1P[8] live oral RV vaccine. OBJECTIVE: To determine the genotypes of RV co-circulating in a non-vaccinated population, in northern Portugal in the winter season of 2007. STUDY DESIGN: Prospective multicenter study of the genotypes circulating in the northwest region of Portugal during January to March 2007. Children with acute gastroenteritis, who attended the Pediatric Emergency Services of five Hospitals, were included in the study. The parents of the children completed a clinical and epidemiological data questionnaire and stool samples were collected. Stool samples positive in a RV enzyme immunoassay (EIA) were genotyped by reverse transcriptase-polymerase chain reaction. RESULTS: Stool samples were collected from 424 children. Two hundred and thirty-four (55.2%) stool samples were RV-positive. G2P[4] was the predominant RV type (68.6%), followed by G9P[8] (14.0%). CONCLUSIONS: Because our population was naïve for RV vaccine, the G2P[4] predominance cannot be explained by vaccination. Rather, this high prevalence of G2P[4] may be within the normal fluctuation of RV genotypes. RV strain surveillance programmes are important for informing RV vaccination programmes.