Microbiological diagnosis of pleural infections: a comparative evaluation of a novel syndromic real-time PCR panel.

Current microbial diagnostics for pleural infections are insufficient. Studies using 16S targeted next-generation sequencing report that only 10%-16% of bacteria present are cultured and that 50%-78% of pleural fluids containing relevant microbial DNA remain culture negative. As a rapid diagnostic alternative suitable for clinical laboratories, we wanted to explore a PCR-based approach. Based on the identification of key pathogens, we developed a syndromic PCR panel for community-acquired pleural infections (CAPIs). This was a pragmatic PCR panel, meaning that it was not designed for detecting all possibly involved bacterial species but for confirming the diagnosis of CAPI, and for detecting bacteria that might influence choice of antimicrobial treatment. We evaluated the PCR panel on 109 confirmed CAPIs previously characterized using culture and 16S targeted next-generation sequencing. The PCR secured the diagnosis of CAPI in 107/109 (98.2%) and detected all present pathogens in 69/109 (63.3%). Culture secured the diagnosis in 54/109 (49.5%) and detected all pathogens in 31/109 (28.4%). Corresponding results for 16S targeted next-generation sequencing were 109/109 (100%) and 98/109 (89.9%). For bacterial species included in the PCR panel, PCR had a sensitivity of 99.5% (184/185), culture of 21.6% (40/185), and 16S targeted next-generation sequencing of 92.4% (171/185). None of the bacterial species present not covered by the PCR panel were judged to impact antimicrobial therapy. A syndromic PCR panel represents a rapid and sensitive alternative to current diagnostic approaches for the microbiological diagnosis of CAPI.IMPORTANCEPleural empyema is a severe infection with high mortality and increasing incidence. Long hospital admissions and long courses of antimicrobial treatment drive healthcare and ecological costs. Current methods for microbiological diagnostics of pleural infections are inadequate. Recent studies using 16S targeted next-generation sequencing as a reference standard find culture to recover only 10%-16% of bacteria present and that 50%-78% of samples containing relevant bacterial DNA remain culture negative. To confirm the diagnosis of pleural infection and define optimal antimicrobial therapy while limiting unnecessary use of broad-spectrum antibiotics, there is a need for rapid and sensitive diagnostic approaches. PCR is a rapid method well suited for clinical laboratories. In this paper we show that a novel syndromic PCR panel can secure the diagnosis of pleural infection and detect all bacteria relevant for choice of antimicrobial treatment with a high sensitivity.

Nausea Predicts Bacteremia in Immunocompetent Patients with Pneumococcal Community-Acquired Pneumonia: Secondary Data Analysis from a Prospective Cohort.

BACKGROUND: In pneumococcal community-acquired pneumonia (CAP), bacteremia is associated with increased mortality, but initial clinical severity scores frequently fail to identify bacteremic patients at risk. We have previously shown that gastrointestinal symptoms are common among patients admitted to the hospital with pneumococcal bacteremia. The aim of this study was to examine gastrointestinal symptoms and inflammatory responses in bacteremic and non-bacteremic pneumococcal CAP in a prospective cohort of immunocompromised and immunocompetent patients hospitalized with CAP. METHODS: Logistic regression analysis was used to estimate the predictive value of gastrointestinal symptoms for pneumococcal bacteremia in patients with CAP. The Mann-Whitney test was used to compare inflammatory responses in patients with bacteremic vs. non-bacteremic pneumococcal CAP. RESULTS: Eighty-one patients with pneumococcal CAP were included, of whom 21 (26%) had bacteremia. Immunocompetent patients with pneumococcal CAP had an odds ratio of 16.5 (95% CI 3.0-90.9, p = 0.001) for bacteremia if nausea was present, whereas no such association was found in the immunocompromised patients (OR 0.22, 95% CI 0.02-2.05, p = 0.18). The serum levels of C-reactive protein, procalcitonin and interleukin 6 were significantly higher in the patients with bacteremic pneumococcal CAP compared to non-bacteremic pneumococcal CAP patients (p < 0.001, p = 0.005, and p = 0.019, respectively). CONCLUSIONS: In immunocompetent patients hospitalized with pneumococcal CAP, nausea may be a predictor of bacteremia. Bacteremic pneumococcal CAP patients display an increased inflammatory response compared to non-bacteremic pneumococcal CAP patients.

Pleural Empyema Caused by Streptococcus intermedius and Fusobacterium nucleatum: A Distinct Entity of Pleural Infections.

BACKGROUND: Many community-acquired pleural infections are caused by facultative and anaerobic bacteria from the human oral microbiota. The epidemiology, clinical characteristics, pathogenesis, and etiology of such infections are little studied. The aim of the present prospective multicenter cohort study was to provide a thorough microbiological and clinical characterization of such oral-type pleural infections and to improve our understanding of the underlying etiology and associated risk factors. METHODS: Over a 2-year period, we included 77 patients with community-acquired pleural infection, whereof 63 (82%) represented oral-type pleural infections. Clinical and anamnestic data were systematically collected, and patients were offered a dental assessment by an oral surgeon. Microbial characterizations were done using next-generation sequencing. Obtained bacterial profiles were compared with microbiology data from previous investigations on odontogenic infections, bacteremia after extraction of infected teeth, and community-acquired brain abscesses. RESULTS: From the oral-type pleural infections, we made 267 bacterial identifications representing 89 different species. Streptococcus intermedius and/or Fusobacterium nucleatum were identified as a dominant component in all infections. We found a high prevalence of dental infections among patients with oral-type pleural infection and demonstrate substantial similarities between the microbiology of such pleural infections and that of odontogenic infections, odontogenic bacteremia, and community-acquired brain abscesses. CONCLUSIONS: Oral-type pleural infection is the most common type of community-acquired pleural infection. Current evidence supports hematogenous seeding of bacteria from a dental focus as the most important underlying etiology. Streptococcus intermedius and Fusobacterium nucleatum most likely represent key pathogens necessary for establishing the infection.

Host Transcriptional Signatures Predict Etiology in Community-Acquired Pneumonia: Potential Antibiotic Stewardship Tools.

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.

Hepcidin predicts 5-year mortality after community-acquired pneumonia.

BACKGROUND: Virtually all living organisms, including microbes and humans, depend on iron to survive and grow. During an infection, the plasma level of iron and several iron-related proteins change substantially. We hypothesized that iron and iron-related proteins could predict short- and long-term outcomes in community-acquired pneumonia. METHODS: Blood samples from a prospective cohort of 267 in-patients with community-acquired pneumonia were analysed for hepcidin, ferritin, iron, transferrin, transferrin saturation, and soluble transferrin receptor at admission and 6-weeks post-discharge. Adverse short-term outcome was defined as admission to intensive care unit or death within 30 days, and long-term outcome was assessed as 5-year overall mortality. Logistic regression, Kaplan Meier survival curves, and Cox regression models with cut-offs at median for the potential biomarkers were used for statistical evaluation. RESULTS: Low admission levels of hepcidin predicted 5-year overall mortality, independently of age, sex, comorbid conditions, and anaemia. Low levels of ferritin at admission as well as low levels of iron and transferrin saturation and high levels of soluble transferrin receptor at the 6-week follow-up were predictors of 5-year overall mortality in univariable, but not in multivariable analyses. Neither of these potential biomarkers predicted adverse short-term outcomes. CONCLUSIONS: In hospitalized patients with community-acquired pneumonia, low levels of hepcidin at admission predicted 5-year overall mortality, but not short-term adverse outcome.

Hepcidin and Ferritin Predict Microbial Etiology in Community-Acquired Pneumonia.

BACKGROUND: Iron is crucial for survival and growth of microbes. Consequently, limiting iron availability is a human antimicrobial defense mechanism. We explored iron and iron-related proteins as potential biomarkers in community-acquired pneumonia and hypothesized that infection-induced changes in these potential biomarkers differ between groups of pathogens and could predict microbial etiology. METHODS: Blood samples from a prospective cohort of 267 patients with community-acquired pneumonia were analyzed for hepcidin, ferritin, iron, transferrin, and soluble transferrin receptor at admission, clinical stabilization, and a 6-week follow-up. A total of 111 patients with an established microbiological diagnosis confined to 1 microbial group (atypical bacterial, typical bacterial, or viral) were included in predictive analyses. RESULTS: High admission levels of ferritin predicted atypical bacterial versus typical bacterial etiology (odds ratio [OR], 2.26; 95% confidence interval [CI], 1.18-4.32; P = .014). Furthermore, hepcidin and ferritin predicted atypical bacterial versus viral etiology (hepcidin: OR = 3.12, 95% CI = 1.34-7.28, P = .008; ferritin: OR = 2.38, 95% CI = 1.28-4.45, P = .006). The findings were independent of C-reactive protein and procalcitonin. CONCLUSIONS: Hepcidin and ferritin are potential biomarkers of microbial etiology in community-acquired pneumonia.

Inflammatory biomarkers are associated with aetiology and predict outcomes in community-acquired pneumonia: results of a 5-year follow-up cohort study.

BACKGROUND: Biomarkers may facilitate clinical decisions in order to guide antimicrobial treatment and prediction of prognosis in community-acquired pneumonia (CAP). We measured serum C-reactive protein, procalcitonin (PCT) and calprotectin levels, and plasma pentraxin 3 (PTX3) and presepsin levels, along with whole-blood white cell counts, at three time-points, and examined their association with microbial aetiology and adverse clinical outcomes in CAP. METHODS: Blood samples were obtained at hospital admission, clinical stabilisation and 6-week follow-up from 267 hospitalised adults with CAP. Adverse short-term outcome was defined as intensive care unit admission and 30-day mortality. Long-term outcome was evaluated as 5-year all-cause mortality. RESULTS: Peak levels of all biomarkers were seen at hospital admission. Increased admission levels of C-reactive protein, PCT and calprotectin were associated with bacterial aetiology of CAP, while increased admission levels of PCT, PTX3 and presepsin were associated with adverse short-term outcome. In univariate and multivariate regression models, white blood cells and calprotectin at 6-week follow-up were predictors of 5-year all-cause mortality. CONCLUSIONS: Calprotectin emerges as both a potential early marker of bacterial aetiology and a predictor for 5-year all-cause mortality in CAP, whereas PCT, PTX3 and presepsin may predict short-term outcome.

Low Levels of Immunoglobulins and Mannose-Binding Lectin Are Not Associated With Etiology, Severity, or Outcome in Community-Acquired Pneumonia.

BACKGROUND: Disease severity and outcome in community-acquired pneumonia (CAP) depend on the host and on the challenge of the causal microorganism(s). We measured levels of immunoglobulins (Igs) and complement in 257 hospitalized adults with CAP and examined the association of low levels of Igs or complement to microbial etiology, disease severity, and short-term and long-term outcome. METHODS: Serum Igs were analyzed in blood samples obtained at admission and at 6 weeks postdischarge if admission levels were low. Serum complement deficiencies were screened with a total complement activity enzyme-linked immunosorbent assay (ELISA), with further analyzes performed if justified. Disease severity was assessed by the CURB-65 severity score. Short-term outcome was defined as a composite end point of intensive care unit (ICU) admission and 30-day mortality, and long-term outcome as 5-year all-cause mortality. RESULTS: At admission, 87 (34%) patients had low levels of at least 1 Ig, with low IgG2 as the most prevalent finding (55/21%). IgG levels were lower in bacterial than viral CAP (8.48 vs 9.97 g/L, P = .023), but low Igs were not associated with microbial etiology. Fifty-five (21%) patients had low lectin pathway activity, of which 33 (13%) were mannose-binding lectin (MBL) deficient. Low admission levels of any Ig or MBL were not associated with disease severity, short-term outcome, or long-term outcome. Excluding patients defined as immunocompromised from analysis did not substantially affect these results. CONCLUSION: In hospitalized adults with CAP, low admission levels of Igs or complement were in general not associated with microbial etiology, disease severity, short-term outcome, or long-term outcome.

Cytokine responses, microbial aetiology and short-term outcome in community-acquired pneumonia.

BACKGROUND: The inflammatory response to community-acquired pneumonia (CAP) is orchestrated through activation of cytokine networks and the complement system. We examined the association of multiple cytokines and the terminal complement complex (TCC) with microbial aetiology, disease severity and short-term outcome. MATERIALS AND METHODS: Plasma levels of 27 cytokines and TCC were analysed in blood samples obtained at hospital admission, clinical stabilization and 6-week follow-up from 247 hospitalized adults with CAP. Fourteen mediators were included in final analyses. Adverse short-term outcome was defined as intensive care unit (ICU) admission and 30-day mortality. RESULTS: Cytokine and TCC levels were dynamic in the clinical course of CAP, with highest levels seen at admission for most mediators. Admission levels of cytokines and TCC did not differ between groups of microbial aetiology. High admission levels of IL-6 (odds ratio [OR] 1.47, 95% confidence interval [CI] 1.18-1.84, P = .001), IL-8 (OR 1.79, 95% CI 1.26-2.55, P = .001) and MIP-1ß (OR 2.28, 95% CI 1.36-3.81, P = .002) were associated with a CURB-65 severity score of ≥3, while IL-6 (OR 1.37, 95% CI 1.07-1.74, P = .011) and MIP-1ß (OR 1.86, 95% CI 1.03-3.36, P = .040) were associated with a high risk of an adverse short-term outcome. CONCLUSIONS: In this CAP cohort, admission levels of IL-6, IL-8 and MIP-1ß were associated with disease severity and/or adverse short-term outcome. Still, for most mediators, only nonsignificant variations in inflammatory responses were observed for groups of microbial aetiology, disease severity and short-term outcome.