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.

www.nature.com/scientificreports/ reports on the incorporation of syndromic tests in clinical practice are scarce, and there is limited evidence of the clinical impact of these tests. Our primary objective was to explore the feasibility of introducing the BioFire FilmArray Pneumonia panel plus (FAP plus) (bioMérieux S.A., Marcy-l'Etoile, France) in the diagnostic workup of patients admitted with suspected CAP, with a view to inform the design of a subsequent randomised controlled trial. We hypothesized that syndromic PCR-based testing of lower respiratory tract samples is achievable in acute settings. The secondary objectives were to investigate the real-world turnaround time (TAT) and microbiological yield of the FAP plus compared to SDs at our hospital.

Methods
Patients and study design. This was a prospectively recruited cohort study conducted at Haukeland University Hospital, a tertiary care referral centre in Bergen, Norway between December 2nd 2019, and February 17th 2020. In addition to conventional microbiological diagnostics, samples from the lower respiratory tract were systematically analysed with a commercial rapid syndromic PCR panel, the FAP plus. The study was conducted as a feasibility study to inform the design of a larger randomised controlled trial evaluating the clinical impact of the FAP plus assay on antibiotic use and outcome (NCT04660084). Patients were eligible for inclusion if they were ≥ 18 years, presenting to the emergency department (ED) with a suspicion of CAP (evaluated by investigating physicians and/or study nurses) and fulfilling at least two of the following criteria: new or worsening cough; new or worsening expectoration of sputum; new or worsening dyspnoea; haemoptysis; pleuritic chest pain; radiological evidence of pneumonia; abnormalities on chest auscultation and/or percussion; fever (≥ 38.0 °C). Exclusion criteria were cystic fibrosis, severe bronchiectasis (defined as patients in need of regular follow-up and treatment by a pulmonologist due to bronchiectasis), hospitalisation within the last 14 days prior to admission, a palliative approach (defined as life expectancy below two weeks documented by a treating physician; either by preexisting estimates in the electronic journal, or estimations made at admission), or if the patient was not willing or able to provide a lower respiratory tract sample (by either sputum induction or endotracheal aspiration).
Most patients were enrolled in the ED shortly after admission. Investigating physicians and/or study nurses screened all electronic triage documents. Patients with respiratory complaints and/or a suspected infection of any type were then evaluated for eligibility, according to the inclusion criteria. To compensate for the restricted study operating hours, some cases were included at the wards up to a maximum of 24 h after admission. These patients were identified through retrospective screening of electronic triage documents and medical records. Relevant baseline information was collected by study nurses or investigating physicians through a structured interview. Symptoms and findings upon clinical examinations were recorded. Data pertaining to treatment and results from laboratory tests and medical imaging were obtained from electronic medical records and charts. Data were registered in an electronic case report form (eCRF) from VieDoc (Viedoc Technologies, Uppsala, Sweden).
The final diagnosis (as per the four different categories enumerated below) was determined retrospectively, following patient discharge, by the use of pre-specified diagnostic criteria in consensus meetings among investigating physicians. The diagnostic criteria thus differed from the inclusion criteria, which were designed to be used as a screening tool based on the available information at admission in the ED. The complete definitions are provided in the Supplementary material (S2). Patients were categorized into four different categories: (1) Confirmed CAP (with radiological confirmation); (2) Clinical CAP (without radiological confirmation); (3) Other respiratory tract infections (RTIs), i.e., bronchitis, acute infectious exacerbation in patients with chronic obstructive pulmonary disease (COPD), acute infectious exacerbation of asthma, and upper RTIs; or (4) Other diagnosis, i.e., CAP suspected at admission but RTI later disproved. To avoid observer bias an independent agreement between the treating physicians and the study investigators was desired. If any disagreement, an additional study investigator would arbitrate.
Microbiological sampling and methods. At inclusion, a lower respiratory tract sample for the FAP plus and standard culture was obtained from all patients. Depending on clinical symptoms, vital signs and medical history, sputum induction with either nebulized isotonic (0.9%) or hypertonic (5.8%) saline was attempted. Patients with known obstructive lung disease and patients with hypoxemia or signs of airway obstruction upon physical examination, were additionally treated with a bronchodilator (salbutamol and/or ipratropium bromide) prior to sampling. If sputum induction was unsuccessful, endotracheal aspiration was performed. The detailed procedures are provided in the Supplementary material (S3).
The FAP plus is an automated multiplex PCR test for the detection of 27 bacteria and viruses, as well as seven genetic markers of antibiotic resistance, validated for lower respiratory tract samples (Supplementary material, S1). The hands-on time is around two minutes and the total analysis time about 1 h 20 . Typical bacterial detections are reported in a semi-quantitative manner and categorized as negative if ≤ 10 3.5 copies/ml. Above this level, results are reported as positive and semi-quantitatively specified as 10 4 , 10 5 , 10 6 or ≥ 10 7 copies/ml 21 .
The SDs included culture of respiratory tract samples and blood according to current guidelines (adapted from 22 ). Blood culture isolates and relevant respiratory isolates were identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) using the Bruker's microflex LT instrument, MBT Compass software ver. 4.1 and Compass Library DB-8468 (Bruker Daltonics, Massachusetts, U.S.). Nasopharyngeal and/or oropharyngeal swabs were examined by an in-house real-time PCR test to detect respiratory viruses and atypical bacteria (influenza A and B, human parainfluenza viruses 1-3, respiratory syncytial virus, . Any additional tests requested by the treating physician were also noted and counted as part of SDs. Gram staining was utilized to evaluate the representativeness of all sputum samples (adapted from 22 ). Samples containing ≥ 10 squamous epithelial cells per field in at least 10 fields with 10 × enlargement were considered non-representative. However, this criterion was disregarded if a significant amount of both leukocytes (≥ 10 times the amount of squamous epithelial cells per field of view) and a morphologically uniform microbe (> 5 microbes per field of view with 100× enlargement) were present. All samples were analysed by the FAP plus and cultured on agar-plates, irrespective of the representativeness. Abundant growth of plausible respiratory pathogens was reported regardless of the representativeness of the sputum sample. Non-abundant growth was only reported in samples considered representative.
The clinical relevance of all microbiological findings, in terms of categorization as a relevant pathogen for the current RTI or not, was established retrospectively using pre-specified criteria (Supplementary material, S4).

Ethics. The study was approved by the Regional Committee for Medical and Health Research Ethics in
South-Eastern Norway (REK ID: 31935) and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants or from their legal guardian/close relative at the time of recruitment.
Respiratory tract specimens were obtained in all RTI-patients, mainly by sputum induction (91/96 (95%)), the remaining by endotracheal aspiration (5/96 (5%)). By Gram-staining, 56 of 96 (58%) respiratory tract samples were classified as not representative of the lower respiratory tract. All samples were investigated using the FAP plus rapid PCR panel in addition to standard culture-based methods. Blood cultures were performed in 95 (99%), in-house PCR testing in 87 (91%), the POC influenza test in 76 (79%), and a pneumococcal urine antigen test in 62 (65%) of the patients.
Time to results. The TAT, i.e., time from sampling to a reported test result, varied considerably by method (

Discussion
This prospective study is one of the first to evaluate the real-world performance and time to results of a rapid syndromic PCR panel in patients presenting to hospital with suspected CAP. We demonstrated that the routine collection of lower respiratory tract specimens (induced sputum or endotracheal aspirates) in the ED was feasible and found a large improvement in both time to microbiological results and the microbiological yield by use of FAP plus compared to SDs. Study enrolment and collection of specimens were in general performed before medical imaging and laboratory results were available. Our patient population is therefore confined to suspected rather than confirmed CAP patients, with the rationale to maximize the potential impact on initial diagnostics and treatment decisions, and reflect actual clinical practice. During the last decade, research on the etiology of CAP has often included PCR-based methods, although usually limited to a restricted selection of bacterial targets or specimens from the upper respiratory tract 3,6,23,24 . One of the most comprehensive studies is a British investigation of sputum from 323 patients with CAP 8 . Like our results, a pathogen was identified in 87% of patients representing a substantial improvement compared to www.nature.com/scientificreports/ standard diagnostic testing (30-40%) 3,7 . However, the British study analysed frozen samples retrospectively with resource-consuming in-house PCR procedures. A short analysis time and ease of use are major advantages of automated commercial PCR panels. When embedded in routine clinical practice, we observed a real-world   Table 2). A direct consequence of a rapid TAT is the provision of near real-time information to treating physicians. This study thus demonstrates a promising potential for informed initial decisions on antimicrobial treatment and isolation. Still, further exploration is needed, preferably in a larger randomised controlled trial, before implementation as a routine test can be considered. The most frequent pathogens in our CAP cohort were H. influenzae and S. pneumoniae, a finding similar to that in other studies 6,8,17,[24][25][26][27] . In Norway, monotherapy with benzylpenicillin, 1.2 g every six hours, is the current empirical treatment recommendation for mild and moderate hospitalised CAP patients. The high prevalence of H. influenzae is consistent with previous PCR-based studies and needs further exploration, moreover, EUCAST has stated that there is still insufficient data for H. influenzae to set clinical breakpoints for benzylpenicillin 28 . Viral pathogens were also frequently detected in our CAP cohort, in agreement with other studies influenced Table 3. Microbiological findings by use of the BioFire FilmArray Pneumonia panel plus versus standard microbiological methods in 72 patients with CAP. Microbiological findings provided by the syndromic PCR panel (FAP plus) compared to SDs in CAP patients (n = 72). Detections deemed as clinically relevant a pathogens are further specified. *Data are shown as number of detections with percentage of the respective microbe's total detections (All methods combined) in brackets. **Data are shown as number of relevant detections with percentage of the respective microbe's total relevant detections (All methods combined) in brackets. Statistically significant differences (McNemar's test, p < 0.05) between the FAP plus and SDs, are marked with bold fonts. CAP, community acquired pneumonia; FAP plus, Biomérieux BioFire FilmArray Pneumonia panel plus; SDs, standard diagnostic methods; -, not applicable; FIA, fluorescent immunoassay. a The clinical relevance of all detected microbes was evaluated retrospectively using pre-specified criteria (Supplementary material, S4). b Sputum culture detected five patients with S. pneumoniae. A pneumococcal antigen detection test in urine (Sofia S. Pneumoniae FIA, Quidel) was positive in ten patients, of which five were unique findings, whereas one was in combination with a positive blood culture. c Detected in blood culture only. d Legionella pneumophila antigen detection test in urine (Sofia Legionella FIA, Quidel).  www.nature.com/scientificreports/ by winter enrolment 6,23 . The growing recognition of an important role for viruses in CAP is thus supported by our work 3,[9][10][11][12][13] . Samples from the lower respiratory tract are often difficult to obtain in a clinically meaningful time frame. Previously reported collection rates of sputum range from 30 to 60% 3,6,13,23 and this represents a major barrier to introducing rapid molecular diagnostics for CAP. Induced sputum was obtained from the majority of included patients (95%) in this study, underscoring that successful collection of this material is feasible, well tolerated and achievable in an ED setting. By traditional microscopic criteria, 42% of specimens were considered representative, a rate not different from other reports 3,6,23 . These microscopic criteria have been developed as a pragmatic quality check of samples in traditional culture-based diagnostics. Modern PCR methods can detect and quantify potentially pathogenic bacteria from complex background microbial populations. In this context, the value of preanalytic microscopy remains uncertain 29 . Detections of viruses and atypical bacteria in RTI-patients are generally considered pathogenic 3,10,12,[30][31][32] . Some data supports that detection of higher quantities of bacterial pathogens are rare among adults without RTI 10,13,31,32 ; but this is disputed, especially in patients with chronic obstructive pulmonary disease 33,34 . Intended to aid in the differentiation between colonising bacteria and causative agents,    www.nature.com/scientificreports/ the FAP plus provides semi-quantitative information on copy numbers for the bacterial pathobionts. Like others, we chose not to consider semi-quantitative values in our study, because of limited evidence from prior use, variable impact on antimicrobial treatment, and the possible influence of different comorbidities on patterns of colonisation and susceptibility to infections 18 . The correlation between semi-quantitative bacterial PCR levels and clinical relevance requires further exploration in larger settings. Recent studies reporting on the performance of FAP plus also show a considerable increase in bacterial detections compared to SDs [14][15][16][17][18][19] . However, important limitations include heterogeneous patient populations (random combinations of community-, hospital-, and/or ventilator acquired pneumonia, as well as other RTIs), various sample materials (sputum, tracheal aspirates or broncho-alveolar lavage) and inconsistent testing by SDs [14][15][16][17]19 . Clinical data are often scarce, and incompletely accounted for. Therefore, the relevance of many of the detected microbes is uncertain. Evaluation of the clinical relevance of all microbiological results is thus a major strength of this study. Only one other recent FAP plus study in CAP patients has made an attempt to determine potential colonisers 18 . Unlike our study, their algorithm included an evaluation of procalcitonin levels to determine relevance and category of infection. The utility of procalcitonin to distinguish viral from bacterial infections is, however, uncertain 35 . Anyhow, determining relevance is difficult, and although not being causative per se, microbes of uncertain relevance could still contribute to an ongoing infectious process. Information about these microbes could therefore be valuable in the case of antibiotic treatment failure.
The pragmatic nature of this study i.e., embedded in routine clinical care hospital practice, the prospective enrolment of patients with community acquired RTIs, application of a stringent case definition of CAP and the analysis of rigorously collected, homogeneous lower respiratory tract samples are major strengths of our work. The study has limitations; the inclusion of a limited number of CAP patients and a short recruitment window during the winter months, suggesting that the findings should be confirmed in further studies. Moreover, patients that were unable to provide a sample from the lower respiratory tract were excluded; resulting in the most severe cases of CAP not being represented, which in turn could explain the low in-hospital mortality rate observed in this study (1%) compared to similar settings 36 .
In conclusion, the use of a rapid syndromic PCR panel for respiratory pathogens was associated with a dramatically reduced time to actionable results and increased detection of clinically relevant pathogens, compared to SDs. A stepwise algorithm for sampling of respiratory specimens with induced sputum and tracheal aspiration was feasible in routine clinical practice and led to obtaining a lower respiratory tract sample in the majority of patients. This suggests that syndromic PCR-based testing may be feasible in acute settings and holds potential to provide clinically actionable results in near real-time. The clinical impact of rapid syndromic pneumonia panels on antibiotic use and patient outcome should therefore be urgently assessed in randomised controlled trials.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.