Genomic Insights into the expansion of Carbapenem-Resistant Klebsiella pneumoniae within Portuguese Hospitals.

Carbapenem-resistant Klebsiella pneumoniae complex (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal CR-KP rate has increased steeply, but the factors associated to this expansion are poorly explored. To address this question we compared, by phylogenetic and resistome analysis, the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. We found that CR-KP belonged mainly to ST13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by KPC-3 (74%) or OXA-181 (18%) presence, which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multidrug resistant harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infection isolates were highly related and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified, by cgMLST data analysis (<5 alleles), 41 occurred between different hospitals and 130 within the same hospital. Our results suggest that CR-KP dissemination in the Lisbon region result from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients and transmission of CR-KP within and between hospitals. Our data reinforces that the prudent use of carbapenems, patients screening at hospital entrance, and improvement of infection control will be needed to decrease the burden of CR-KP infection in Portugal.

A COVID-19 Outbreak in a Rheumatology Department Upon the Early Days of the Pandemic.

Objectives: To describe our experience with a coronavirus disease 2019 (COVID-19) outbreak within a large rheumatology department early in the pandemic. Methods: Symptomatic and asymptomatic healthcare workers (HCWs) had a naso-oropharyngeal swab for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and were followed clinically. Reverse transcription polymerase-chain reaction (RT-PCR) was repeated to document cure, and serological response was assessed. Patients with risk contacts within the department in the 14 days preceding the outbreak were screened for COVID-19 symptoms. Results: 14/34 HCWs (41%; 40 ± 14 years, 71% female) tested positive for SARS-CoV-2, and 11/34 (32%) developed symptoms but were RT-PCR-negative. Half of RT-PCR-positive HCWs did not report fever, cough, or dyspnea before testing, which were absent in 3/14 cases (21%). Mild disease prevailed (79%), but 3 HCWs had moderate disease requiring further assessment, which excluded severe complications. Nevertheless, symptom duration (28 ± 18 days), viral shedding (31 ± 10 days post-symptom onset, range 15-51), and work absence (29 ± 28 days) were prolonged. 13/14 (93%) of RT-PCR-positive and none of the RT-PCR-negative HCWs had a positive humoral response Higher IgG indexes were observed in individuals over 50 years of age (14.5 ± 7.7 vs. 5.0 ± 4.4, p = 0.012). Of 617 rheumatic patients, 8 (1.3%) developed COVID-19 symptoms (1/8 hospitalization, 8/8 complete recovery), following a consultation/procedure with an asymptomatic (7/8) or mildly symptomatic (1/8) HCW. Conclusions: A COVID-19 outbreak can occur among HCWs and rheumatic patients, swiftly spreading over the presymptomatic stage. Mild disease without typical symptoms should be recognized and may evolve with delayed viral shedding, prolonged recovery, and adequate immune response in most individuals.

Relevancia a corto plazo de la coinfección viral en pacientes menores de 2 años hospitalizados con infecciones de las vías respiratorias inferiores / Short-term relevance of lower respiratory viral coinfection in inpatients under 2 years of age

Introducción: Avances en el diagnóstico molecular han hecho posible la detección de agentes virales desconocidos en infecciones de las vías respiratorias inferiores (IVRI). Sin embargo, sigue habiendo dudas relativamente a su frecuencia y relevancia. Objetivo: Comparar la clínica y la gravedad entre la infección por virus único y la coinfección en niños admitidos por IVRI. Métodos: Se realizó un estudio durante 3 años consecutivos (2012-2015) que incluyó a niños menores de 2 años ingresados por IVRI. La identificación viral se realizó mediante la técnica de PCR para 16 virus. Los datos clínicos y el uso de los recursos hospitalarios se recogieron de forma estándar durante la estancia hospitalaria y se compararon la infección única con coinfecciones virales. Resultados: Fueron analizadas 524 muestras (451 pacientes); 448 (85,5%) tuvieron al menos un virus identificado. Coinfecciones virales se encontraron en 159 (35,5%). RSV y HRV fueron los virus más frecuentes; bronquiolitis y neumonía, los diagnósticos principales. Los pacientes con coinfecciones virales eran mayores, iban a la guardería, tenían sibilancias recurrentes con más frecuencia y eran más sintomáticos al ingreso. No fueron sometidos a más exámenes, pero les fueron prescritos medicamentos con más frecuencia. El grupo de la coinfección viral no mostró una mayor duración de la estancia hospitalaria, de la necesidad de oxígeno, de UCI o soporte ventilatorio. Discusión: Nuestro estudio mostró una proporción significativa de coinfecciones virales en los niños pequeños ingresados con IVRI y confirma dados previos que muestran que la prescripción es más frecuente en las coinfecciones virales, sin asociación con peor resultado clínico (AU)

Introduction: Advances in molecular diagnosis have made it possible to detect previously unknown viral agents as causative agents of lower respiratory tract infections (LRTI). The frequency and relevance of viral coinfections is still debatable. Objective: compare clinical presentation and severity between single virus infection and viral coinfection in children admitted for LRTI. Methods: A 3-year period observational study (2012-2015) included children younger than two years admitted for LRTI. Viral identification was performed using PCR technique for 16 viruses. Clinical data and use of health resources was gathered during hospital stay using a standard collection form and we compared single virus infection and viral coinfections. Results: The study included 524 samples (451 patients); 448 (85,5%) had at least one virus identified. Viral coinfections were found in 159 (35,5%). RSV and HRV were the most commonly identified virus; bronchiolitis and pneumonia the most frequent diagnosis. Patients with viral coinfections were older, attended day-care centers, had previous recurrent wheezing more frequently and were more symptomatic at admission. These patients did not have more complementary exams performed but were prescribed medications more often. Viral coinfection group did not show longer length of hospital stay and oxygen need, more need for ICU nor ventilatory support. Discussion: Our study showed a significant proportion of viral coinfections in young infants admitted with LRTI and confirmed previous data showing that prescription was more frequent in inpatients with viral coinfections, without an association with worst clinical outcome (AU)

[Short-term relevance of lower respiratory viral coinfection in inpatients under 2 years of age]. / Relevancia a corto plazo de la coinfección viral en pacientes menores de 2 años hospitalizados con infecciones de las vías respiratorias inferiores.

INTRODUCTION: Advances in molecular diagnosis have made it possible to detect previously unknown viral agents as causative agents of lower respiratory tract infections (LRTI). The frequency and relevance of viral coinfections is still debatable. OBJECTIVE: compare clinical presentation and severity between single virus infection and viral coinfection in children admitted for LRTI. METHODS: A 3-year period observational study (2012-2015) included children younger than two years admitted for LRTI. Viral identification was performed using PCR technique for 16 viruses. Clinical data and use of health resources was gathered during hospital stay using a standard collection form and we compared single virus infection and viral coinfections. RESULTS: The study included 524 samples (451 patients); 448 (85,5%) had at least one virus identified. Viral coinfections were found in 159 (35,5%). RSV and HRV were the most commonly identified virus; bronchiolitis and pneumonia the most frequent diagnosis. Patients with viral coinfections were older, attended day-care centers, had previous recurrent wheezing more frequently and were more symptomatic at admission. These patients did not have more complementary exams performed but were prescribed medications more often. Viral coinfection group did not show longer length of hospital stay and oxygen need, more need for ICU nor ventilatory support. DISCUSSION: Our study showed a significant proportion of viral coinfections in young infants admitted with LRTI and confirmed previous data showing that prescription was more frequent in inpatients with viral coinfections, without an association with worst clinical outcome.

Polymicrobial biofilms by diabetic foot clinical isolates.

Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is foot ulceration that may be colonized by pathogenic and antimicrobial resistant bacteria, which may express several virulence factors that could impair treatment success. These bacterial communities can be organized in polymicrobial biofilms, which may be responsible for diabetic foot ulcer (DFU) chronicity. We evaluated the influence of polymicrobial communities in the ability of DFU isolates to produce biofilm, using a microtiter plate assay and a multiplex fluorescent in situ hybridization, at three time points (24, 48, 72 h), after evaluating biofilm formation by 95 DFU isolates belonging to several bacterial genera (Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas and Acinetobacter). All isolates were biofilm-positive at 24 h, and the amount of biofilm produced increased with incubation time. Pseudomonas presented the higher biofilm production, followed by Corynebacterium, Acinetobacter, Staphylococcus and Enterococcus. Significant differences were found in biofilm formation between the three time points. Polymicrobial communities produced higher biofilm values than individual species. Pseudomonas + Enterococcus, Acinetobacter + Staphylococcus and Corynebacterium + Staphylococcus produced higher biofilm than the ones formed by E. faecalis + Staphylococcus and E. faecalis + Corynebacterium. Synergy between bacteria present in dual or multispecies biofilms has been described, and this work represents the first report on time course of biofilm formation by polymicrobial communities from DFUs including several species. The biological behavior of different bacterial species in polymicrobial biofilms has important clinical implications for the successful treatment of these infections.

Use of flow cytometry (Sysmex) UF-100) to screen for positive urine cultures: in search for the ideal cut-off.

BACKGROUND: Cultures for urinary tract infections (UTI) constitute a large workload in the clinical microbiology laboratory, although up to 80% are usually negative. Several automated methods are available to screen urines for UTI, one being the flow cytometry-based Sysmex((R)) UF-100. METHODS: The performance of the UF-100 was evaluated over a 16-month period using urine culture as the reference method. RESULTS: During this period, a total of 5356 urine samples were studied (469 children; 3229 women and 1658 men), of which 706 were culture positive (593 grew Gram negative bacilli). Receiver operating characteristics (ROC) curve analysis showed an area under the curve (AUC) of 0.83 for leukocytes and 0.85 for bacterial count. Applying cut-off values reported in the literature gave sensitivities ranging from 75% to 90%, resulting in 73-174 false negatives (FN). Using a logical combination (leukocytes >or=15x10(6)/L OR bacteria >or=500x10(6)/L) gave a sensitivity of 98%. However, the specificity dropped to 25%, resulting in 15 FN. CONCLUSIONS: Screening urine samples for UTI detects a large number of culture positive samples. However, the rather large number of FN observed precludes the use of the UF-100 as a routine screening method to exclude urine samples from culture.

Survey of extended-spectrum beta-lactamases in Escherichia coli isolates from a Portuguese hospital and characterisation of a novel class 1 integron (In60A) carrying the blaCTX-M-9 gene.

Between November 2001 and November 2004, 231 Escherichia coli isolates resistant to beta-lactam antibiotics were identified. In 14 isolates, bla(TEM-24) (2 isolates), bla(TEM-52) (5 isolates) and bla(TEM-26) (7 isolates) were identified. In 145 E. coli isolates with the same M13 fingerprinting profile and the same resistance phenotype, the bla(CTX-M-15) gene was found in association with an insertion sequence ISEcp1. The bla(CTX-M-2) gene was identified in one E. coli isolate (290HSM), and in other E. coli isolate (246HSM) the bla(CTX-M-9) gene was contained in a new complex sul1-type class 1 integron (named In60A). This is the first report of three cefotaximases (CTX-M-15, CTX-M-2 and CTX-M-9) in E. coli isolates from a Portuguese hospital.