Rapid syndromic PCR testing in patients with respiratory tract infections reduces time to results and improves microbial yield.

Lack of rapid and comprehensive microbiological diagnosis in patients with community acquired pneumonia (CAP) hampers appropriate antimicrobial therapy. This study evaluates the real-world performance of the BioFire FilmArray Pneumonia panel plus (FAP plus) and explores the feasibility of evaluation in a randomised controlled trial. Patients presenting to hospital with suspected CAP were recruited in a prospective feasibility study. An induced sputum or an endotracheal aspirate was obtained from all participants. The FAP plus turnaround time (TAT) and microbiological yield were compared with standard diagnostic methods (SDs). 96/104 (92%) enrolled patients had a respiratory tract infection (RTI); 72 CAP and 24 other RTIs. Median TAT was shorter for the FAP plus, compared with in-house PCR (2.6 vs 24.1 h, p < 0.001) and sputum cultures (2.6 vs 57.5 h, p < 0.001). The total microbiological yield by the FAP plus was higher compared to SDs (91% (162/179) vs 55% (99/179), p < 0.0001). Haemophilus influenzae, Streptococcus pneumoniae and influenza A virus were the most frequent pathogens. In conclusion, molecular panel testing in adults with CAP was associated with a significant reduction in time to actionable results and increased microbiological yield. The impact on antibiotic use and patient outcome should be assessed in randomised controlled trials.

The bacterial aetiology of pleural empyema. A descriptive and comparative metagenomic study.

OBJECTIVES: The view of pleural empyema as a complication of bacterial pneumonia is changing because many patients lack evidence of underlying pneumonia. To further our understanding of pathophysiological mechanisms, we conducted in-depth microbiological characterization of empyemas in clinically well-characterized patients and investigated observed microbial parallels between pleural empyemas and brain abscesses. METHODS: Culture-positive and/or 16S rRNA gene PCR-positive pleural fluids were analysed using massive parallel sequencing of the 16S rRNA and rpoB genes. Clinical details were evaluated by medical record review. Comparative analysis with brain abscesses was performed using metagenomic data from a national Norwegian study. RESULTS: Sixty-four individuals with empyema were included. Thirty-seven had a well-defined microbial aetiology, while 27, all of whom had community-acquired infections, did not. In the latter subset, Fusobacterium nucleatum and/or Streptococcus intermedius was detected in 26 patients, of which 18 had additional facultative and/or anaerobic species in various combinations. For this group, there was 65.5% species overlap with brain abscesses; predisposing factors included dental infection, minor chest trauma, chronic obstructive pulmonary disease, drug abuse, alcoholism and diabetes mellitus. Altogether, massive parallel sequencing yielded 385 bacterial detections, whereas culture detected 38 (10%) and 16S rRNA gene PCR/Sanger-based sequencing detected 87 (23%). CONCLUSIONS: A subgroup of pleural empyema appears to be caused by a set of bacteria not normally considered to be involved in pneumonia. Such empyemas appear to have a similar microbial profile to oral/sinus-derived brain abscesses, supporting spread from the oral cavity, potentially haematogenously. We suggest reserving the term 'primary empyema' for these infections.

Deciphering the aetiology of a mixed fungal infection by broad-range PCR with sequencing and fluorescence in situ hybridisation.

Simultaneous infections with multiple fungi may be misinterpreted as monomicrobial infections by current diagnostics with ramifications for the choice of antimicrobial agents that may impact patient outcomes. The application of molecular methods on tissue samples may be useful to decipher the aetiology of mixed fungal infections. We present a leukaemic patient who died from sepsis due to candidaemia. The postmortem examination documented fungal elements in lung tissue. Fungal DNA was amplified from the lung sample by broad-range PCR assays targeting the 28S ribosomal RNA gene or the internal transcribed spacer 2 (ITS-2). Fluorescence in situ hybridisation (FISH) using differentially labelled fungal probes was applied on the tissue. Sequencing identified the PCR amplicons as Aspergillus fumigatus (28S assay) and Candida tropicalis (ITS-2 assay). As a chromatogram suggested mixed amplicons, the Isentio ripseq(®) tool for in silico analysis was applied and confirmed the presence of both amplicons in the PCR products of both assays. FISH confirmed the presence of Aspergillus and Candida within the infectious process, a prerequisite for inferring a causal relationship with the infection. The combination of broad-range PCR with sequence analysis and FISH applied on tissue samples is a powerful approach to identify the aetiology of invasive fungal infections, including mixed infections.