The role of time to positive blood cultures in enhancing the predictive capability of DENOVA score for diagnosing infective endocarditis in patients with Enterococcus faecalis bacteremia.

DENOVA-score is useful to stratify the risk of infective endocarditis (IE) in Enterococcus faecalis bacteremia. Recently, time to positive (TTP) of blood cultures has also been related with a higher risk of IE. The objective was to evaluate DENOVA- score with TTP to improve its specificity. We performed a retrospective, case-control study in adult patients with E. faecalis bacteremia. Thirty-nine patients with definite E. faecalis IE and 82 with E. faecalis bacteremia were included. The addition of a TTP ≤ 8 h to DENOVA-score did not improve the diagnostic accuracy of this score.

Case report: BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream the spike 69/70 deletion.

BACKGROUND: SARS-CoV-2 variant tracking is key to the genomic surveillance of the COVID-19 pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification. CASE PRESENTATION: A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike amino acids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation. CONCLUSIONS: To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the probe target area of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.

Streptococcus downii sp. nov., isolated from the oral cavity of a teenager with Down syndrome.

A new α-haemolytic streptococcal strain has been isolated from the dental plaque of a teenager with Down syndrome. Genetic and taxonomic analyses place this Streptococcus within the oralis group. It is a Gram-stain-positive, non-motile, non-spore-forming spherical alpha-haemolytic coccus arranged in chains, and it ferments a large number of monosaccharides and disaccharides, as well as polymeric carbohydrates. It differs biochemically from closely related species of Streptococcus due to its production of α-galactosidase, ß-galactosidase and N-acetyl-ß-d-glucosaminidase and by the absence of arginine dihydrolase deiminase and IgA1-protease. It grows in a temperature range of 25 to 40 °C (optimal growth temperature at 37 °C) and in a pH range of 4.5 to 8 (optimal pH at 7.0). A phylogenetic analysis based on its 16S and 23S rRNA gene sequences placed it close to Streptococcus dentisani CECT 7747T. The ANIb and ANIm values were 93.19 and 93.61 %, respectively, both below the accepted threshold to designate it as a new species of bacteria. A phylogenetic tree based on its core genome placed it close to Streptococcus oralis subsp. dentisani strain CECT 7747T with a distance in the expanded core phylogeny of 0.1298. The in silico DNA-DNA hybridization value was 52.2 % with respect to the closest species, S. oralis subsp. dentisani CECT 7747T. Based on these data, a new species of bacteria within the genus Streptococcus, family Streptococcaceae and order Lactobacillales is described, for which the name of Streptococcus downii sp. nov. is proposed (type strain CECT 9732T=CCUG 73139T).

Pooling for SARS-CoV-2 control in care institutions.

BACKGROUND: Workers and residents in Care Homes are considered at special risk for the acquisition of SARS-CoV-2 infection, due to the infectivity and high mortality rate in the case of residents, compared to other containment areas. The role of presymptomatic people in transmission has been shown to be important and the early detection of these people is critical for the control of new outbreaks. Pooling strategies have proven to preserve SARS-CoV-2 testing resources. The aims of the present study, based in our local experience, were (a) to describe SARS-CoV-2 prevalence in institutionalized people in Galicia (Spain) during the Coronavirus pandemic and (b) to evaluate the expected performance of a pooling strategy using RT-PCR for the next rounds of screening of institutionalized people. METHODS: A total of 25,386 Nasopharyngeal swab samples from the total of the residents and workers at Care Homes in Galicia (March to May 2020) were individually tested using RT-PCR. Prevalence and quantification cycle (Cq) value distribution of positives was calculated. Besides, 26 pools of 20 samples and 14 pools of 5 samples were tested using RT-PCR as well (1 positive/pool). Pooling proof of concept was performed in two populations with 1.7 and 2% prevalence. RESULTS: Distribution of SARS-CoV-2 infection at Care Homes was uneven (0-60%). As the virus circulation global rate was low in our area (3.32%), the number of people at risk of acquiring the infection continues to be very high. In this work, we have successfully demonstrated that pooling of different groups of samples at low prevalence clusters, can be done with a small average delay on Cq values (5 and 2.85 cycles for pools of 20 and 5 samples, respectively). CONCLUSIONS: A new screening system with guaranteed protection is required for small clusters, previously covered with individual testing. Our proposal for Care Homes, once prevalence zero is achieved, would include successive rounds of testing using a pooling solution for transmission control preserving testing resources. Scale-up of this method may be of utility to confront larger clusters to avoid the viral circulation and keeping them operative.

Moving beyond unsolicited consultation: additional impact of a structured intervention on mortality in Staphylococcus aureus bacteraemia.

BACKGROUND: Some evidence-based bundles have tried to standardize the management of Staphylococcus aureus bacteraemia (SAB) to improve the outcome. The aim of our study was to analyse the additional impact on mortality of a structured intervention in patients with SAB. METHODS: Compliance with the bundle was evaluated in an ambispective cohort of patients with SAB, which included a retrospective cohort [including patients treated before and after the implementation of a bacteraemia programme (no-BP and BP, respectively)] and a prospective cohort (i-BP), in which an additional specific intervention for bundle application was implemented. Multivariate logistic regression was used to measure the influence of the independent variables including compliance with the bundle on 14 and 30 day crude mortality. RESULTS: A total of 271 adult patients with SAB were included. Mortality was significantly different among the three groups (no-BP, BP and i-BP): mortality at 14 days was 18% versus 7% versus 2%, respectively, P = 0.002; and mortality at 30 days was 20% versus 12% versus 5%, respectively, P = 0.011. The factors associated with 14 and 30 day mortality in multivariable analysis were heart failure (OR = 7.63 and OR = 2.27, respectively), MRSA infection (OR = 4.02 and OR = 4.37, respectively) and persistent bacteraemia (OR = 11.01 and OR = 7.83, respectively); protective factors were catheter-related bacteraemia (OR = 0.16 and OR = 0.19, respectively) and >75% bundle compliance (OR = 0.15 and OR = 0.199, respectively). Time required to perform the intervention and the follow-up was 50 min (IQR 40-55 min) per patient. CONCLUSIONS: High-level compliance with a standardized bundle of intervention for management of SAB that requires little time was associated with lower mortality at 14 and 30 days.

Short- versus long-course therapy in gram-negative bacilli bloodstream infections.

Bacteremia due to gram-negative bacilli (GNB) is one of the most frequent causes of admission and prolongation of hospital stay. Nevertheless, optimal duration of antibiotic treatment is not clearly established. We designed an observational, prospective study of a cohort of adult patients with uncomplicated GNB bacteremia. They were classified according to the duration of treatment in short (7-10 days) or long (> 10 days) course. Mortality and bacteremia recurrence rate were evaluated, and propensity score for receiving short-course treatment was calculated. A total of 395 patients met eligibility criteria including 232 and 163 who received long- and short-course therapy, respectively. Median age was 71 years, 215 (54.4% male) and 51% had a urinary source. Thirty-day mortality was 12%. In multivariate analysis by logistic regression stratified according to propensity score quartile for receiving short-course therapy showed no association of duration of treatment with 30-day mortality or 90-day recurrence rate. Based on the results observed in our cohort, short-course therapy could be as safe and effective as longer courses.

Effectiveness of ceftazidime/avibactam as salvage therapy for treatment of infections due to OXA-48 carbapenemase-producing Enterobacteriaceae.

Background: Experience in real clinical practice with ceftazidime/avibactam is limited, and there are even fewer data on infections due to OXA-48-producing Enterobacteriaceae. Methods: We designed an observational study of a prospectively collected cohort of adult patients receiving ceftazidime/avibactam in our centre. Only the first treatment course of each patient was analysed. Efficacy and safety were evaluated as 14 and 30 day mortality, recurrence rate at 90 days, resistance development and occurrence of adverse effects. Results: Fifty-seven patients were treated with ceftazidime/avibactam. The median age was 64 years (range 26-86), 77% were male and the median Charlson index was 3. The most frequent sources of infection were intra-abdominal (28%), followed by respiratory (26%) and urinary (25%). Thirty-one (54%) patients had a severe infection (defined as presence of sepsis or septic shock). Most patients received ceftazidime/avibactam as monotherapy (81%) and the median duration of treatment was 13 days. Mortality at 14 days was 14%. In multivariate analysis, the only mortality risk factor was INCREMENT-CPE score >7 (HR 11.7, 95% CI 4.2-20.6). There was no association between mortality and monotherapy with ceftazidime/avibactam. The recurrence rate at 90 days was 10%. Ceftazidime/avibactam resistance was not detected in any case and only two patients developed adverse events related to treatment. Conclusions: Ceftazidime/avibactam shows promising results, even in monotherapy, for the treatment of patients with severe infections due to OXA-48-producing Enterobacteriaceae and limited therapeutic options. The emergence of resistance to ceftazidime/avibactam was not observed.

[New clone of ST-187 Acinetobacter baumannii responsible for an outbreak in an intensive care unit]. / Nuevo clon de Acinetobacter baumannii ST187 responsable de un brote en una unidad de cuidados intensivos.

INTRODUCTION: An ICU-outbreak caused by a novel Acinetobacter baumannii clone is described. METHODS: An active search of carriers and environmental reservoirs was carried out. Carbapenemases genes were studied using multiplex-PCR and genotypic analysis by rep-PCR, PFGE and MLST. RESULTS: A total 26 infected patients and 10 carriers were identified. A.baumannii was recovered from infusion pumps, walls, floor and washbasins. Phenotypic/genotypic analysis showed clonal expansion of a unique clone ST-187 producer of type OXA-24 and OXA-51 carbapenemases. DISCUSSION: This is the first outbreak caused by ST-187 (ECI/GCI) multiresistant A.baumannii.

Comparison of dalbavancin with standard of care in the management of infective endocarditis: efficacy, safety, and cost analysis.

OBJECTIVES: Due to its long half-life, dalbavancin offers benefits for long-duration treatments, especially osteoarticular and infective endocarditis (IE). We evaluated the efficacy and costs of IE treatment, comparing dalbavancin with standard of care (SOC). METHODS: Retrospective multicenter cohort study of adult patients with Gram-positive cocci definite IE. Dalbavancin was used as a sequential therapy before discharge. Efficacy was a combined variable of clinical cure and absence of recurrence in 12-month follow-up. Length of hospital stay and the associated costs were analyzed in both groups of treatment. RESULTS: Twenty-two patients received dalbavancin and 47 SOC. The efficacy was similar between the groups (dalbavancin 18 [72%] vs SOC 44 [94%], P = 0.198). Hospital stay was shorter in the dalbavancin group (dalbavancin 22 days [16-34] vs SOC 37 days [23-49], P = 0.001), especially in those with E. faecalis IE (dalbavancin 30 days [20-36] vs SOC 65 days [46-74], P <0.001). A reduction of cost was observed between both groups (dalbavancin, 12,206 € [8998-17,283] vs SOC 16,249 € [11,496-22,367], P = 0.032). CONCLUSION: Dalbavancin could be a safe and effective option in the sequential treatment of patients with IE. Also, a cost reduction was detected, due to a significant shortness of hospital stay.

Efficacy and safety of a structured de-escalation from antipseudomonal ß-lactams in bloodstream infections due to Enterobacterales (SIMPLIFY): an open-label, multicentre, randomised trial.

BACKGROUND: De-escalation from broad-spectrum to narrow-spectrum antibiotics is considered an important measure to reduce the selective pressure of antibiotics, but a scarcity of adequate evidence is a barrier to its implementation. We aimed to determine whether de-escalation from an antipseudomonal ß-lactam to a narrower-spectrum drug was non-inferior to continuing the antipseudomonal drug in patients with Enterobacterales bacteraemia. METHODS: An open-label, pragmatic, randomised trial was performed in 21 Spanish hospitals. Patients with bacteraemia caused by Enterobacterales susceptible to one of the de-escalation options and treated empirically with an antipseudomonal ß-lactam were eligible. Patients were randomly assigned (1:1; stratified by urinary source) to de-escalate to ampicillin, trimethoprim-sulfamethoxazole (urinary tract infections only), cefuroxime, cefotaxime or ceftriaxone, amoxicillin-clavulanic acid, ciprofloxacin, or ertapenem in that order according to susceptibility (de-escalation group), or to continue with the empiric antipseudomonal ß-lactam (control group). Oral switching was allowed in both groups. The primary outcome was clinical cure 3-5 days after end of treatment in the modified intention-to-treat (mITT) population, formed of patients who received at least one dose of study drug. Safety was assessed in all participants. Non-inferiority was declared when the lower bound of the 95% CI of the absolute difference in cure rate was above the -10% non-inferiority margin. This trial is registered with EudraCT (2015-004219-19) and ClinicalTrials.gov (NCT02795949) and is complete. FINDINGS: 2030 patients were screened between Oct 5, 2016, and Jan 23, 2020, of whom 171 were randomly assigned to the de-escalation group and 173 to the control group. 164 (50%) patients in the de-escalation group and 167 (50%) in the control group were included in the mITT population. 148 (90%) patients in the de-escalation group and 148 (89%) in the control group had clinical cure (risk difference 1·6 percentage points, 95% CI -5·0 to 8·2). The number of adverse events reported was 219 in the de-escalation group and 175 in the control group, of these, 53 (24%) in the de-escalation group and 56 (32%) in the control group were considered severe. Seven (5%) of 164 patients in the de-escalation group and nine (6%) of 167 patients in the control group died during the 60-day follow-up. There were no treatment-related deaths. INTERPRETATION: De-escalation from an antipseudomonal ß-lactam in Enterobacterales bacteraemia following a predefined rule was non-inferior to continuing the empiric antipseudomonal drug. These results support de-escalation in this setting. FUNDING: Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases; Spanish Clinical Research and Clinical Trials Platform, co-financed by the EU; European Development Regional Fund "A way to achieve Europe", Operative Program Intelligence Growth 2014-2020.

Mechanism of Action of Streptococcus downii, a New Bacterial Species with Probiotic Potential.

Streptococcus downii is a recently reported bacterial species of oral origin, with inhibitory capacity against Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula and Aggregatibacter actinomycetemcomitans, which confers upon it the potential of being an oral probiotic. The aim of the present study was to identify the potential mechanisms by which S. downii exerts its inhibitory effect on S. mutans. To this end, the study assessed the consumption of glucose and proteins available in the culture medium, the modification of the pH, the production of short-chain fatty acids, the changes in the protein panel of the inhibition halo, the production of hydrogen peroxide and the effect of proteinase K. There were no differences in the glucose values or in the protein content of the medium, but there was a reduction in pH (with no effect on the growth of S. mutans). Significant increases were detected in the levels of lactic and formic acid (with no effect on the growth of S. mutans), as well as changes in the peptide panel (with no effect on the growth of S. mutans). The inhibitory effect was maintained in the presence of peroxidase but disappeared after adding proteinase K. Based on these results, it is suggested that the main mechanism of inhibition of S. downii against S. mutans is the production of bacteriocins.

Proof of concept of the potential of a machine learning algorithm to extract new information from conventional SARS-CoV-2 rRT-PCR results.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been and remains one of the major challenges modern society has faced thus far. Over the past few months, large amounts of information have been collected that are only now beginning to be assimilated. In the present work, the existence of residual information in the massive numbers of rRT-PCRs that tested positive out of the almost half a million tests that were performed during the pandemic is investigated. This residual information is believed to be highly related to a pattern in the number of cycles that are necessary to detect positive samples as such. Thus, a database of more than 20,000 positive samples was collected, and two supervised classification algorithms (a support vector machine and a neural network) were trained to temporally locate each sample based solely and exclusively on the number of cycles determined in the rRT-PCR of each individual. Overall, this study suggests that there is valuable residual information in the rRT-PCR positive samples that can be used to identify patterns in the development of the SARS-CoV-2 pandemic. The successful application of supervised classification algorithms to detect these patterns demonstrates the potential of machine learning techniques to aid in understanding the spread of the virus and its variants.

Molecular Detection of Streptococcus downii sp. nov. from Dental Plaque Samples from Patients with Down Syndrome and Non-Syndromic Individuals.

A new bacterial species has recently been identified in the dental plaque of an adolescent with Down syndrome. The species is known as Streptococcus downii sp. nov. (abbreviated to S. downii), and it inhibits the growth of S. mutans and certain periodontal pathogens. The aim of this study was to determine the distribution of S. downii in the oral cavity of individuals with Down syndrome. Methods: A specific polymerase chain reaction for the operon of bacteriocin (class IIb lactobin A/cerein 7B family) was designed to detect S. downii in individuals with Down syndrome (n = 200) and in the general population (n = 100). We also compared the whole genome of S. downii and the regions related to its bacteriocins against 127 metagenomes of supragingival plaque of the "Human Microbiome Project". Results: We detected the specific gene of the S. downii bacteriocin in an individual with Down syndrome (Cq, 34.52; GE/µL, 13.0) and in an individual of the non-syndromic control group (Cq, 34.78 Cq; GE/µL, 4.93). The prevalence of S. downii was ≤1% both in Down syndrome and in the general population, which did not allow for clinical-microbiological correlations to be established. This result was confirmed by detecting only one metagenome with an ANIm with approximately 95% homology and with 100% homology with ORFs that code class IIb lactobiocin A/cerein 7B bacteriocins among the 127 metagenomes of the "Human Microbiome Project" tested. Conclusions: The detection rate of S. downii in the supragingival dental plaque was very low, both in the Down syndrome individuals and in the non-syndromic controls. A clinical-microbiological correlation could therefore not be established.

[Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry vs conventional methods in the identification of Candida non-albicans]. / Espectrometría de masas matrix-assisted laser desorption ionization time-of-flight vs. metodología convencional en la identificación de Candida no-albicans.

INTRODUCTION: Candida infection has become a major health problem worldwide. The epidemiology of Candidaemia has substantially changed by the emergence of the species Candida non-albicans. This variation is particularly important in the choice of prophylaxis and empirical treatment. The methods based on biochemical and molecular biology have limitations for the correct identification of Candida species. The aim of this study is to demonstrate the ability of the MALDI-TOF mass spectrometry for the identification of these species and compare it with the technology used today. METHODS: We included all isolates collected over 2 years (n=73) of Candida non-albicans from non-invasive samples. The identification was carried out by Vitek-2 systems YST and API CAUX. The MALDI-TOF identifications were made with Confidence Axima system (Shimadzu Corporation) using the Shimadzu Launchpad software and database SARAMIS (AnagnosTec GmbH). Discrepancies were resolved by SeptiFast LightCycler multiplex PCR, specific PCR C. glabrata and enzymatic digestion with BanI SADH fragment in isolates of C. parapsilosis. RESULTS: Of the 73 isolates of Candida non-albicans, the biochemical methods conclusively identified 67 to species level and 6 at the genus level. The MALDI-TOF system obtained identifications at the species level in all cases. The correlation in the species of all isolates studied was 85.07%, reaching 94.52% when the correlation was made between the identification obtained by biochemical methods and the methods for the analysis of the discrepancies. In isolates of C. parapsilosis, MALDI-TOF system obtained an identification of C. orthopsilosis. In 3 of them it was confirmed by digestion with BanI SADH fragment. CONCLUSION: This study has demonstrated the use of mass spectrometry (MALDI-TOF system) to provide the microbiology laboratory with greater efficiency and reliability to identify isolates of Candida non-albicans to species level. It also shows its potential usefulness in identifying related species, such as C. parapsilosis, metapsilosis and orthopsilosis.

In Vitro Anti-Biofilm and Antibacterial Properties of Streptococcus downii sp. nov.

The aim of this study was to evaluate the potential anti-biofilm and antibacterial activities of Streptococcus downii sp. nov. To test anti-biofilm properties, Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were grown in a biofilm model in the presence or not of S. downii sp. nov. for up to 120 h. For the potential antibacterial activity, 24 h-biofilms were exposed to S. downii sp. nov for 24 and 48 h. Biofilms structures and bacterial viability were studied by microscopy, and the effect in bacterial load by quantitative polymerase chain reaction. A generalized linear model was constructed, and results were considered as statistically significant at p < 0.05. The presence of S. downii sp. nov. during biofilm development did not affect the structure of the community, but an anti-biofilm effect against S. mutans was observed (p < 0.001, after 96 and 120 h). For antibacterial activity, after 24 h of exposure to S. downii sp. nov., counts of S. mutans (p = 0.019) and A. actinomycetemcomitans (p = 0.020) were significantly reduced in well-structured biofilms. Although moderate, anti-biofilm and antibacterial activities of S. downii sp. nov. against oral bacteria, including some periodontal pathogens, were demonstrated in an in vitro biofilm model.

The fate of SARS-COV-2 in WWTPS points out the sludge line as a suitable spot for detection of COVID-19.

SARS-CoV-2 genetic material is detectable in the faeces of a considerable part of COVID-19 cases and hence, in municipal wastewater. This fact was confirmed early during the spread of the COVID-19 pandemic and prompted several studies that proposed monitoring its incidence by wastewater. This paper studies the fate of SARS-CoV-2 genetic material in wastewater treatment plants using RT-qPCR with a two-fold goal: i) to check its presence in the water effluent and in the produced sludge and ii) based on the understanding of the virus particles fate, to identify the most suitable spots for detecting the incidence of COVID-19 and monitor its evolution. On the grounds of the affinity of enveloped virus towards biosolids, we hypothesized that the sludge line acts as a concentrator of SARS-CoV-2 genetic material. Sampling several spots in primary, secondary and sludge treatment at the Ourense (Spain) WWTP in 5 different days showed that, in effect, most of SARS-CoV-2 particles cannot be detected in the water effluent as they are retained by the sludge line. We identified the sludge thickener as a suitable spot for detecting SARS-CoV-2 particles thanks to its higher solids concentration (more virus particles) and longer residence time (less sensitive to dilution caused by precipitation). These findings could be useful to develop a suitable strategy for early warning of COVID-19 incidence based on WWTP monitoring.