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Sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes throughout the COVID-19 pandemic has generated a wealth of data on viral evolution across populations, but only a few studies have so far explored SARS-CoV-2 evolution across large connected transmission networks. Here, we couple data from SARS-CoV-2 sequencing with contact tracing data from an outbreak with a single origin in a rural Norwegian community where samples from all exposed persons were collected prospectively. A total of 134 nasopharyngeal samples were positive by PCR. Among the 121 retrievable genomes, 81 were identical to the genome of the introductor, thus demonstrating that genomics beyond clustering genotypically similar viral genomes to confirm relatedness offers limited additional value to manual contact tracing. In the cases where mutations were discovered, five small genetic clusters were identified. We observed a household secondary attack rate of 77%, with 92% of household members infected among households with secondary transmission, suggesting that SARS-CoV-2 introduction into large families is likely to affect all household members. IMPORTANCE: In outbreak investigations, obtaining a full overview of infected individuals within a population is seldom achieved. We here present an example where a single introduction of B1.1.7 SARS-CoV-2 within a rural community allowed for tracing of the virus from an introductor via dissemination through larger gatherings into households. The outbreak occurred before widespread vaccination, allowing for a "natural" outbreak development with community lockdown. We show through sequencing that the virus can infect up to five consecutive persons without gaining mutations, thereby showing that contact tracing seems more important than sequencing for local outbreak investigations in settings with few alternative introductory transmission pathways. We also show how larger households where a child introduced transmission appeared more likely to promote further spread of the virus compared to households with an adult as the primary introductor.
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BACKGROUND: Hospital-acquired pneumonia (HAP) is the most common hospital-acquired infection (HAI). HAP is associated with a high burden of morbidity and mortality, but the diagnosis is difficult to establish and the incidence uncertain. METHODS: Patients aged ≥ 18 years hospitalised with radiologically verified non-ventilator hospital acquired pneumonia (NV-HAP) during 2018 were retrospectively identified at Drammen Hospital, a Norwegian general hospital. Infectious Diseases Society of America and the American Thoracic Society's definition of HAP was used. RESULTS: In total 119 cases of NV-HAP were identified among 27,701 admissions. The incidence was 4.3 per 1000 admissions and 1.2 per 1000 patient-days. The median age was 74 years, 63% were male and median Charlson comorbidity index was 5. Coronary heart disease (42%) was the most common comorbidity. Median length of stay was 17.2 days. A blood culture was obtained in 53.8% of patients, while samples from lower airways were seldom obtained (10.9%). In-hospital mortality was 21%, accumulated 30-day mortality was 27.7% and accumulated 1-year mortality was 39.5%. Thirty-day readmission rate among survivors was 39.4%. CONCLUSION: NV-HAP was present in approximately 1 in 250 hospitalisations, most had multiple comorbidities, and 1 in 5 died in hospital. Although thorough microbiological sampling is recommended when NV-HAP is suspected, our data indicate that airway sampling is infrequent in clinical practice. Our findings underscore the need to develop microbiological diagnostic strategies to achieve targeted antimicrobial treatment that may improve patient outcomes and reduce broad-spectrum antibiotic usage.
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OBJECTIVES: This study aimed to describe the microbial aetiology of community-acquired pneumonia (CAP) in adults admitted to a tertiary care hospital and assess the impact of syndromic polymerase chain reaction (PCR) panels on pathogen detection. METHODS: Conducted at Haukeland University Hospital, Norway, from September 2020 to April 2023, this prospective study enrolled adults with suspected CAP. We analysed lower respiratory tract samples using both standard-of-care tests and the BIOFIRE® FILMARRAY® Pneumonia Plus Panel (FAP plus). The added value of FAP Plus in enhancing the detection of clinically relevant pathogens, alongside standard-of-care diagnostics, was assessed. RESULTS: Of the 3238 patients screened, 640 met the inclusion criteria, with 384 confirmed to have CAP at discharge. In these patients, pathogens with proven or probable clinical significance were identified in 312 (81.3%) patients. Haemophilus influenzae was the most prevalent pathogen, found in 118 patients (30.7%), followed by SARS-CoV-2 in 74 (19.3%), and Streptococcus pneumoniae in 64 (16.7%). Respiratory viruses were detected in 186 (48.4%) patients. The use of FAP plus improved the pathogen detection rate from 62.8% with standard-of-care methods to 81.3%. CONCLUSIONS: Pathogens were identified in 81% of CAP patients, with Haemophilus influenzae and respiratory viruses being the most frequently detected pathogens. The addition of the FAP plus panel, markedly improved pathogen detection rates compared to standard-of-care diagnostics alone.
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Infecções Comunitárias Adquiridas , Humanos , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/epidemiologia , Estudos Prospectivos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Noruega/epidemiologia , Hospitalização , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Pneumonia/microbiologia , Pneumonia/diagnóstico , Idoso de 80 Anos ou mais , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/genética , Haemophilus influenzae/isolamento & purificação , Haemophilus influenzae/genética , Reação em Cadeia da Polimerase/métodos , COVID-19/diagnósticoRESUMO
Importance: Lower respiratory tract (LRT) infections, including community-acquired pneumonia (CAP), are a leading cause of hospital admissions and mortality. Molecular tests have the potential to optimize treatment decisions and management of CAP, but limited evidence exists to support their routine use. Objective: To determine whether the judicious use of a syndromic polymerase chain reaction (PCR)-based panel for rapid testing of CAP in the emergency department (ED) leads to faster, more accurate microbiological test result-based treatment. Design, Setting, and Participants: This parallel-arm, single-blinded, single-center, randomized clinical superiority trial was conducted between September 25, 2020, and June 21, 2022, in the ED of Haukeland University Hospital, a large tertiary care hospital in Bergen, Norway. Adult patients who presented to the ED with suspected CAP were recruited. Participants were randomized 1:1 to either the intervention arm or standard-of-care arm. The primary outcomes were analyzed according to the intention-to-treat principle. Intervention: Patients randomized to the intervention arm received rapid syndromic PCR testing (BioFire FilmArray Pneumonia plus Panel; bioMérieux) of LRT samples and standard of care. Patients randomized to the standard-of-care arm received standard microbiological diagnostics alone. Main Outcomes and Measures: The 2 primary outcomes were the provision of pathogen-directed treatment based on a microbiological test result and the time to provision of pathogen-directed treatment (within 48 hours after randomization). Results: There were 374 patients (221 males [59.1%]; median (IQR) age, 72 [60-79] years) included in the trial, with 187 in each treatment arm. Analysis of primary outcomes showed that 66 patients (35.3%) in the intervention arm and 25 (13.4%) in the standard-of-care arm received pathogen-directed treatment, corresponding to a reduction in absolute risk of 21.9 (95% CI, 13.5-30.3) percentage points and an odds ratio for the intervention arm of 3.53 (95% CI, 2.13-6.02; P < .001). The median (IQR) time to provision of pathogen-directed treatment within 48 hours was 34.5 (31.6-37.3) hours in the intervention arm and 43.8 (42.0-45.6) hours in the standard-of-care arm (mean difference, -9.4 hours; 95% CI, -12.7 to -6.0 hours; P < .001). The corresponding hazard ratio for intervention compared with standard of care was 3.08 (95% CI, 1.95-4.89). Findings remained significant after adjustment for season. Conclusions and Relevance: Results of this randomized clinical trial indicated that routine deployment of PCR testing for LRT pathogens led to faster and more targeted microbial treatment for patients with suspected CAP. Rapid molecular testing could complement or replace selected standard, time-consuming, laboratory-based diagnostics. Trial Registration: ClinicalTrials.gov Identifier: NCT04660084.
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Infecções Comunitárias Adquiridas , Pneumonia , Infecções Respiratórias , Idoso , Humanos , Masculino , Infecções Comunitárias Adquiridas/diagnóstico , Serviço Hospitalar de Emergência , Hospitalização , Pneumonia/diagnóstico , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: Serum neurofilament light chain (sNfL), an indicator of neuronal damage, is increasingly recognized as a potential biomarker for disease activity in neurodegenerative disorders. In this study, we wanted to investigate sNfL as a prognostic marker in a large, well-defined population of 90 patients with Lyme neuroborreliosis (LNB). In addition, we sought to explore associations between symptoms and sNfL levels during the acute phase of LNB. MATERIALS AND METHODS: Patients diagnosed with definite or possible LNB were recruited from a double-blinded, placebo-controlled, multi-center trial, in which the participants were randomly assigned to 2 or 6 weeks of oral doxycycline treatment. The sNfL levels were measured using a single molecule array assay at both diagnosis and 6-month follow-up, and analysed against clinical parameters, variations in symptom burden and long-term complaints as assessed by a composite clinical score. RESULTS: At the time of diagnosis, approximately 60% of the patients had elevated sNfL levels adjusted for age. Notably, mean sNfL levels were significantly higher at diagnosis (52 pg/ml) compared to 6 months after treatment (12 pg/ml, p < 0.001), when sNfL levels had normalized in the majority of patients. Patients with objective signs of spinal radiculitis had significantly higher baseline sNfL levels compared to patients without spinal radiculitis (p = 0.033). CONCLUSION: Our findings suggest that sNfL can serve as a biomarker for peripheral nerve tissue involvement in the acute phase of LNB. As found in an earlier study, we confirm normalization of sNfL levels in blood after treatment. We found no prognostic value of acute-phase sNfL levels on patient outcome.
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Biomarcadores , Neuroborreliose de Lyme , Proteínas de Neurofilamentos , Humanos , Neuroborreliose de Lyme/sangue , Neuroborreliose de Lyme/tratamento farmacológico , Neuroborreliose de Lyme/diagnóstico , Masculino , Feminino , Proteínas de Neurofilamentos/sangue , Pessoa de Meia-Idade , Noruega , Adulto , Biomarcadores/sangue , Idoso , Estudos Longitudinais , Método Duplo-Cego , Antibacterianos/administração & dosagem , Doxiciclina/administração & dosagem , Estudos de Coortes , Carga de SintomasRESUMO
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) spread around the world during the first part of 2020. The purpose of the study was to assess the prevalence of SARS-CoV-2 infection among patients acutely admitted to the Psychiatric Clinic, Haukeland University Hospital. METHODS: Serum tests to assess for antibodies to SARS-CoV-2 were administered at admission to the clinic together with a questionnaire on symptoms and demographical information. Further information was obtained from the medical records. RESULTS: The cumulative seroprevalence in the 266 participants was 0.75%, the cumulative reported cases in the Norwegian general population was 0.61% at the end of the inclusion period of the study. Twenty-five percent of participants had risk factors for a serious course of COVID-19. There was a low prevalence of cohabitation and only 20% had their main income derived from ordinary salaries (not welfare). CONCLUSION: The prevalence of SARS-CoV-2 infection in a sample of patients acutely admitted to the Psychiatric Clinic, Haukeland University Hospital, was comparable to reported cases in the general population. A possible link to governmental and municipal restrictions, general low workplace participation and cohabitation is discussed.
Seroprevalence of SARS-CoV-2 antibodies is comparable to the general population.Twenty-five percent of patients had elevated risk for a serious course of COVID-19 because of somatic conditions.Fifty-seven percent lived alone, 17% with one other person in the household.Twenty percent had regular salary as the main income source for the last three months before admission.
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COVID-19 , Humanos , COVID-19/epidemiologia , SARS-CoV-2 , Unidade Hospitalar de Psiquiatria , Estudos Prospectivos , Pandemias , Estudos Soroepidemiológicos , Anticorpos Antivirais , Noruega/epidemiologiaRESUMO
BACKGROUND: B cell depletion therapy is highly effective in relapsing-remitting multiple sclerosis (RRMS). However, the precise underlying mechanisms of action for its biological effects in MS have still not been clarified. Epstein-Barr virus (EBV) is a known risk factor for MS and seems to be a prerequisite for disease development. EBV resides latently in the memory B cells, and may not only increase the risk of developing MS, but also contribute to disease activity and disability progression. Therefore, the effects of B cell depletion in MS could be associated with the depletion of EBV-infected cells and the altered immune response to the virus. In this study, we investigate the impact of B cell depletion on the humoral immune response specific to EBV in patients with MS. METHODS: Newly diagnosed, treatment-naïve patients with RRMS were followed up to 18 months after initiation of B-cell depletion therapy in the Overlord-MS study, a phase III trial (NCT04578639). We analyzed serum sampled before treatment and after 3, 6, 12 and 18 months for immunoglobulin γ (IgG) against Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr viral capsid antigen (VCA). We analyzed antibodies to cytomegalovirus (CMV) and total IgG in serum, as controls for viral and overall humoral immunity. The risk allele, HLA-DRB1*15:01, and the protective allele, HLA-A*02:01, were determined in all participants. In addition, polymerase chain reaction (PCR) for circulating EBV-DNA was performed in the first 156 samples drawn. The associations between time on B cell-depletion therapy and serum anti-EBV antibody levels were estimated using linear mixed-effects models. RESULTS: A total of 290 serum samples from 99 patients were available for analysis. After 6, 12 and 18 months, the EBNA1 IgG levels decreased by 12.7 % (95 % CI -18.8 to -6.60, p < 0.001), 12.1 % (95 % CI -19.8 to -3.7, p = 0.006) and 14.6 % (95 % CI to -25.3 to -2.4, p = 0.02) respectively, compared to baseline level. Carriers of the HLA-DRB1*15:01 allele had higher EBNA1 IgG levels at baseline (p = 0.02). The VCA IgG levels significantly increased by 13.7 % (95 % CI 9.4 to 18.1, p < 0.001) after 3 months, compared to baseline, and persisted at this level throughout the follow-up. CMV IgG levels decreased, but to a lesser extent than the decrease of EBNA1 IgG, and total IgG levels decreased during therapy. Circulating EBV-DNA was found in only three of 156 samples from 64 patients. CONCLUSIONS: EBNA1 IgG levels decreased, while VCA IgG levels increased, during B cell depletion therapy. This supports the hypothesis that the mechanism of action for B cell depletion therapy might be mediated by effects on EBV infection, which, in turn, mitigate immune cross-reactivity and disease perpetuation.
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Infecções por Citomegalovirus , Infecções por Vírus Epstein-Barr , Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Anticorpos Antivirais , DNA , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/terapia , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4/genética , Cadeias HLA-DRB1/genética , Esclerose Múltipla Recidivante-Remitente/terapiaRESUMO
This prospective study assessed the value of initial microscopy evaluation of sputum samples submitted for rapid syndromic PCR-based testing. Bacterial detections by the BioFire FilmArray Pneumonia Panel plus in 126 high- and 108 low-quality sputum samples, based on initial microscopy evaluation in samples from patients with lower respiratory tract infections were compared. We found that high-quality samples had a higher proportion of bacterial detections compared to low-quality samples (P = 0.013). This included a higher proportion of detections of bacteria deemed clinically relevant by predefined criteria (70% and 55%, P = 0.016), as well as a higher proportion of detections of Haemophilus influenzae (36% and 20%, P = 0.010). High-quality samples also had more detections of bacteria with high semi-quantitative values. The study found no significant difference between high- and low-quality samples in the proportions of samples with a single species of bacteria detected, samples with a bacteria treated by the clinician, samples with detection of a proven etiology of community-acquired pneumonia by predefined criteria, the number of bacterial species detected, or the detection of Streptococcus pneumoniae, Moraxella catarrhalis, or Staphylococcus aureus. The results showed that 40% (95% CI 35%-47%) of the bacterial detections would have been missed if only high-quality samples were analyzed. This included 41% (27%-56%) of detections of S. pneumoniae, 33% (23%-45%) of detections of H. influenzae, 42% (28%-58%) of detections of S. aureus, and 37% (23%-54%) of detections of M. catarrhalis. These findings suggest that all sputum samples submitted for rapid syndromic PCR testing should be analyzed, regardless of initial microscopy quality assessment. (This study has been registered at ClinicalTrials.gov under registration no. NCT04660084.) IMPORTANCE Microscopic quality assessment of sputum samples was originally designed for sputum culture, and its applicability in today's workflow, which includes syndromic PCR testing, may differ. Addressing this crucial gap, our study emphasizes the need to optimize the use and workflow of syndromic PCR panels, like the BioFire FilmArray Pneumonia plus (FAP plus), in microbiology laboratories. These advanced PCR-based tests offer rapid and comprehensive pathogen detection for respiratory infections, yet their full potential remains uncertain. By comparing bacterial detections in high- and low-quality sputum samples, we underscore the importance of including low-quality samples in testing. Our findings reveal a significant proportion of potentially clinically relevant bacterial detections that would have been missed if only high-quality samples were analyzed. These insights support the efficient implementation of syndromic PCR panels, ultimately enhancing patient care and outcomes.
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Syndromic PCR-based analysis of lower respiratory tract (LRT) samples in patients with community-acquired pneumonia (CAP) improves the bacterial yield and time-to-results compared to culture-based methods. However, obtaining adequate sputum samples can be challenging and is frequently not prioritized in the emergency department (ED). In this study, we assess the concordance of microbiological detections between oropharyngeal- (OP) and LRT samples from patients presenting to the ED with CAP using a syndromic PCR-based respiratory panel [Biofire FilmArray Pneumonia plus (FAP plus)]. Paired OP- and high-quality LRT samples were collected from 103 patients with confirmed CAP, who had been included in a randomized controlled trial (NCT04660084) or a subsequent observational study at Haukeland University Hospital, and analyzed using the FAP plus. The LRT samples were obtained mainly by sputum induction (88%). Using the LRT samples as a reference standard, the positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement for the most common bacterial pathogens in CAP, Streptococcus pneumoniae and Haemophilus influenzae, were 85%, 99% and 95%, and 86%, 98% and 93%, respectively. For Moraxella catarrhalis, the PPA was lower (74%), while the NPA was 100%. For bacteria that are less likely causes of uncomplicated CAP (e.g., Staphylococcus aureus and Enterobacterales) the results were more divergent. In conclusion, the FAP plus detects the most common CAP pathogens S. pneumoniae and H. influenzae from OP samples with high PPAs and excellent NPAs when compared with LRT samples. For these pathogens, the PPAs for OP samples were higher than previous reports for nasopharyngeal samples. This suggests that analysis of OP samples with syndromic PCR panels could represent an alternative approach for rapid microbiological testing in the ED, especially in patients where LRT samples are difficult to obtain. Divergent results for bacteria that are less likely to cause uncomplicated CAP do, however, emphasize the need for clinical evaluation of positive test results.
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Infecções Comunitárias Adquiridas , Pneumonia , Humanos , Pneumonia/diagnóstico , Pneumonia/microbiologia , Streptococcus pneumoniae/genética , Reação em Cadeia da Polimerase , Bactérias/genética , Orofaringe/microbiologia , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/microbiologiaRESUMO
BACKGROUND: The COVID-19 pandemic was met with strict containment measures. We hypothesized that societal infection control measures would impact the number of hospital admissions for respiratory tract infections, as well as, the spectrum of pathogens detected in patients with suspected community acquired pneumonia (CAP). METHODS: This study is based on aggregated surveillance data from electronic health records of patients admitted to the hospitals in Bergen Hospital Trust from January 2017 through June 2021, as well as, two prospective studies of patients with suspected CAP conducted prior to and during the COVID-19 pandemic (pre-COVID cohort versus COVID cohort, respectively). In the prospective cohorts, microbiological detections were ascertained by comprehensive PCR-testing in lower respiratory tract specimens. Mann-Whitney's U test was used to analyse continuous variables. Fisher's exact test was used for analysing categorical data. The number of admissions before and during the outbreak of SARS-CoV-2 was compared using two-sample t-tests on logarithmic transformed values. RESULTS: Admissions for respiratory tract infections declined after the outbreak of SARS-CoV-2 (p < 0.001). The pre-COVID and the COVID cohorts comprised 96 and 80 patients, respectively. The proportion of viruses detected in the COVID cohort was significantly lower compared with the pre-COVID cohort [21% vs 36%, difference of 14%, 95% CI 4% to 26%; p = 0.012], and the proportion of bacterial- and viral co-detections was less than half in the COVID cohort compared with the pre-COVID cohort (19% vs 45%, difference of 26%, 95% CI 13% to 41%; p < 0.001). The proportion of bacteria detected was similar (p = 0.162), however, a difference in the bacterial spectrum was observed in the two cohorts. Haemophilus influenzae was the most frequent bacterial detection in both cohorts, followed by Streptococcus pneumoniae in the pre-COVID and Staphylococcus aureus in the COVID cohort. CONCLUSION: During the first year of the COVID-19 pandemic, the number of admissions with pneumonia and the microbiological detections in patients with suspected CAP, differed from the preceding year. This suggests that infection control measures related to COVID-19 restrictions have an overall and specific impact on respiratory tract infections, beyond reducing the spread of SARS-CoV-2.
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COVID-19 , Infecções Comunitárias Adquiridas , Pneumonia , Infecções Respiratórias , COVID-19/epidemiologia , Infecções Comunitárias Adquiridas/epidemiologia , Humanos , Pandemias , Pneumonia/epidemiologia , Estudos Prospectivos , Infecções Respiratórias/epidemiologia , SARS-CoV-2RESUMO
BACKGROUND: Community-acquired pneumonia (CAP) causes a large burden of disease. Due to difficulties in obtaining representative respiratory samples and insensitive standard microbiological methods, the microbiological aetiology of CAP is difficult to ascertain. With a few exceptions, standard-of-care diagnostics are too slow to influence initial decisions on antimicrobial therapy. The management of CAP is therefore largely based on empirical treatment guidelines. Empiric antimicrobial therapy is often initiated in the primary care setting, affecting diagnostic tests based on conventional bacterial culture in hospitalized patients. Implementing rapid molecular testing may improve both the proportion of positive tests and the time it takes to obtain test results. Both measures are important for initiation of pathogen-targeted antibiotics, involving rapid de-escalation or escalation of treatment, which may improve antimicrobial stewardship and potentially patient outcome. METHODS: Patients presenting to the emergency department of Haukeland University Hospital (HUH) in Bergen, Norway, will be screened for inclusion into a pragmatic randomised controlled trial (RCT). Eligible patients with a suspicion of CAP will be included and randomised to receive either standard-of-care methods (standard microbiological testing) or standard-of-care methods in addition to testing by the rapid and comprehensive real-time multiplex PCR panel, the BioFire® FilmArray® Pneumonia Panel plus (FAP plus) (bioMérieux S.A., Marcy-l'Etoile, France). The results of the FAP plus will be communicated directly to the treating staff within ~2 h of sampling. DISCUSSION: We will examine if rapid use of FAP plus panel in hospitalized patients with suspected CAP can improve both the time to and the proportion of patients receiving pathogen-directed treatment, thereby shortening the exposure to unnecessary antibiotics and the length of hospital admission, compared to the standard-of-care arm. The pragmatic design together with broad inclusion criteria and a straightforward intervention could make our results generalizable to other similar centres. TRIAL REGISTRATION: ClinicalTrials.gov NCT04660084 . Registered on December 9, 2020.
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Anti-Infecciosos , Infecções Comunitárias Adquiridas , Pneumonia , Antibacterianos/uso terapêutico , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/tratamento farmacológico , Humanos , Técnicas de Diagnóstico Molecular , Pneumonia/diagnóstico , Pneumonia/tratamento farmacológicoRESUMO
Background: Current approaches for pathogen identification in community-acquired pneumonia (CAP) remain suboptimal, leaving most patients without a microbiological diagnosis. If better diagnostic tools were available for differentiating between viral and bacterial CAP, unnecessary antibacterial therapy could be avoided in viral CAP patients. Methods: In 156 adults hospitalized with CAP classified to have bacterial, viral, or mixed viral-bacterial infection based on microbiological testing or both microbiological testing and procalcitonin (PCT) levels, we aimed to identify discriminatory host transcriptional signatures in peripheral blood samples acquired at hospital admission, by applying Dual-color-Reverse-Transcriptase-Multiplex-Ligation-dependent-Probe-Amplification (dc-RT MLPA). Results: In patients classified by microbiological testing, a 9-transcript signature showed high accuracy for discriminating bacterial from viral CAP (AUC 0.91, 95% CI 0.85-0.96), while a 10-transcript signature similarly discriminated mixed viral-bacterial from viral CAP (AUC 0.91, 95% CI 0.86-0.96). In patients classified by both microbiological testing and PCT levels, a 13-transcript signature showed excellent accuracy for discriminating bacterial from viral CAP (AUC 1.00, 95% CI 1.00-1.00), while a 7-transcript signature similarly discriminated mixed viral-bacterial from viral CAP (AUC 0.93, 95% CI 0.87-0.98). Conclusion: Our findings support host transcriptional signatures in peripheral blood samples as a potential tool for guiding clinical decision-making and antibiotic stewardship in CAP.
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Descriptions of the small intestinal microbiota are deficient and conflicting. We aimed to get a reliable description of the jejunal bacterial microbiota by investigating samples from two separate jejunal segments collected from the luminal mucosa during surgery. Sixty patients with morbid obesity selected for elective gastric bypass surgery were included in this survey. Samples collected by rubbing a swab against the mucosa of proximal and mid jejunal segments were characterized both quantitatively and qualitatively using a combination of microbial culture, a universal quantitative PCR and 16S deep sequencing. Within the inherent limitations of partial 16S sequencing, bacteria were assigned to the species level. By microbial culture, 53 patients (88.3%) had an estimated bacterial density of < 1600 cfu/ml in both segments whereof 31 (51.7%) were culture negative in both segments corresponding to a bacterial density below 160 cfu/ml. By quantitative PCR, 46 patients (76.7%) had less than 104 bacterial genomes/ml in both segments. The most abundant and frequently identified species by 16S deep sequencing were associated with the oral cavity, most often from the Streptococcus mitis group, the Streptococcus sanguinis group, Granulicatella adiacens/para-adiacens, the Schaalia odontolytica complex and Gemella haemolysans/taiwanensis. In general, few bacterial species were identified per sample and there was a low consistency both between the two investigated segments in each patient and between patients. The jejunal mucosa of fasting obese patients contains relatively few microorganisms and a core microbiota could not be established. The identified microbes are likely representatives of a transient microbiota and there is a high degree of overlap between the most frequently identified species in the jejunum and the recently described ileum core microbiota.
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Bactérias/crescimento & desenvolvimento , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Jejuno/microbiologia , Obesidade Mórbida/microbiologia , Adulto , Idoso , Bactérias/genética , DNA Bacteriano/genética , Feminino , Derivação Gástrica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mucosa Intestinal/cirurgia , Jejuno/cirurgia , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/diagnóstico , Obesidade Mórbida/cirurgia , Reação em Cadeia da Polimerase em Tempo Real , Ribotipagem , Adulto JovemRESUMO
BACKGROUND: Respiratory tract infections (RTIs) caused by contagious viruses are common among patients presenting to the emergency department (ED). Early detection of these viruses can help prevent nosocomial transmission. AIM: To investigate the efficacy of three rapid molecular methods, namely FilmArray® Pneumonia Panel plus (FAP plus), ID NOW™ Influenza A and B 2 (ID NOW2) point-of-care test, and an in-house real-time polymerase chain reaction (RT-PCR) test, to identify patients with viral RTIs requiring isolation in an emergency setting. METHODS: We included a FilmArray® Pneumonia Panel plus in the initial workup of patients with suspected RTIs during a flu season. The RT-PCR and the influenza point-of-care test were performed as part of routine diagnostics, on demand from the treating physicians. We compared viral detections and compared time to positive test results for each method. FINDINGS: The FAP plus significantly reduced the turnaround time and was able to identify 95% patients with potential contagious viral RTI. Routine diagnostics ordered by the treating physician had a turnaround time of a median 22 h and detected 87% of patients with potential contagious viral RTI. In patients that had all three tests, the ID NOW2 detected 62% of patients with influenza. CONCLUSIONS: The FAP plus was able to rapidly and reliably identify patients with potential contagious viral RTIs; its use was feasible in the ED setting. Failing to test patients with viral RTI and using tests with long turnaround time may lead to nosocomial transmission of viral infections and adverse patient outcomes.
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Infecção Hospitalar , Serviços Médicos de Emergência , Influenza Humana , Pneumonia , Infecções Respiratórias , Vírus , Infecção Hospitalar/diagnóstico , Humanos , Influenza Humana/diagnóstico , Técnicas de Diagnóstico Molecular , Estudos Prospectivos , Infecções Respiratórias/diagnóstico , Vírus/genéticaRESUMO
In this article, we investigate patterns of microbial DNA contamination in targeted 16S rRNA amplicon sequencing (16S deep sequencing) and demonstrate how this can be used to filter background bacterial DNA in diagnostic microbiology. We also investigate the importance of sequencing depth. We first determined the patterns of contamination by performing repeat 16S deep sequencing of negative and positive extraction controls. This process identified a few bacterial species dominating across all replicates but also a high intersample variability among low abundance contaminant species in replicates split before PCR amplification. Replicates split after PCR amplification yielded almost identical sequencing results. On the basis of these observations, we suggest using the abundance of the most dominant contaminant species to define a threshold in each clinical sample from where identifications with lower abundances possibly represent contamination. We evaluated this approach by sequencing of a diluted, staggered mock community and of bile samples from 41 patients with acute cholangitis and noninfectious bile duct stenosis. All clinical samples were sequenced twice using different sequencing depths. We were able to demonstrate the following: (i) The high intersample variability between sequencing replicates is caused by events occurring before or during the PCR amplification step. (ii) Knowledge about the most dominant contaminant species can be used to establish sample-specific cutoffs for reliable identifications. (iii) Below the level of the most abundant contaminant, it rapidly becomes very demanding to reliably discriminate between background and true findings. (iv) Adequate sequencing depth can be claimed only when the analysis also picks up background contamination. IMPORTANCE There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples. Reporting a contaminant species as a relevant pathogen may cause unnecessary antibiotic treatment or even falsely classify a noninfectious condition as a bacterial infection. Yet, there are few studies on how to filter contamination in clinical microbiology. Here, we demonstrate that sequencing of extraction controls will not reveal the full spectrum of contaminants that could occur in the associated clinical samples. Only the most abundant contaminant species were consistently detected, and we present how this can be used to set sample specific thresholds for reliable identifications. We believe this work can facilitate the implementation of 16S deep sequencing in diagnostic laboratories. The new data we provide on the patterns of microbial DNA contamination is also important for microbiome research.
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Bactérias/genética , Carga Bacteriana/métodos , Técnicas de Laboratório Clínico/métodos , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Ribossômico 16S/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Bile/microbiologia , Colangite/microbiologia , Técnicas de Laboratório Clínico/normas , Contaminação por DNA , Feminino , Humanos , Masculino , Microbiota/genética , Pessoa de Meia-Idade , Análise de Sequência de DNA , Adulto JovemRESUMO
Since the mass vaccination against swine flu in 2009, new knowledge has changed the basis for the assessment of injury compensation and for further research on delayed sequelae following vaccination.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana , Humanos , Vacinação/efeitos adversosRESUMO
OBJECTIVES: The existing literature on the microbiota of the ileum is inconsistent. To further characterize the microbiota, we analysed samples obtained directly from resected ileums used for urinary diversion after radical cystectomy. METHODS: We included 150 patients with bladder cancer operated on from March 2016 to March 2019. Samples obtained by rubbing a swab against the ileal mucosa 25 cm from the ileocecal valve were cultivated at the local laboratory. Microbial colonies were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). RESULTS: The microbial density of the distal ileum was low. Among our samples, 79% (95% confidence interval (CI) 71%, 84%) harboured less than 1.6 × 104 cfu/mL, whereas 36% (95% CI 28%, 44%) harboured less than 1.6 × 103 cfu/mL. The flora was dominated by viridans streptococci, Candida, Actinomyces, Rothia and Lactobacillus species. Colon-related bacteria i.e. strict anaerobic bacteria, Enterobacteriales and enterococci, were recovered from 14% of the samples. Constipation was associated with increased recovery of colon-related bacteria. Antibiotic treatment prior to surgical procedures did not affect culture results. Increased age was significantly associated with more substantial fungal growth and use of proton pump inhibitors seemed to increase both bacterial and fungal growth. CONCLUSIONS: The microbiota of the human distal ileum is sparse and differs significantly from the colonic microbiota both quantitatively and by composition. These findings contradict recent metagenomics studies based on samples collected by retrograde colonoscopy and emphasize the crucial importance of adequate sampling techniques.