Introduction

Lower respiratory tract infections (LRTIs), including pneumonia, remain a major cause of morbidity and mortality worldwide across all age groups [1]. Prompt identification of causative pathogens is essential for initiating appropriate antimicrobial therapy and improving clinical outcomes. However, conventional culture-based diagnostic methods are limited by prolonged turnaround times, reduced sensitivity following prior antibiotic exposure, and suboptimal detection of fastidious or atypical organisms [2,3].

Pneumonia can be classified as community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), and ventilator-associated pneumonia (VAP), and may be caused by a wide variety of bacterial, viral, and atypical pathogens. The increasing prevalence of multidrug-resistant (MDR) organisms has further complicated empirical treatment strategies, underscoring the importance of rapid detection of antimicrobial resistance (AMR) determinants [2,4]. In addition, conventional phenotypic antimicrobial susceptibility testing generally requires 48–96 hours, often delaying appropriate initiation, escalation, or de-escalation of antimicrobial therapy [5].

Multiplex polymerase chain reaction (PCR)-based syndromic diagnostic platforms have emerged as promising tools to overcome the limitations of conventional microbiological methods. Among these, the BioFire FilmArray Pneumonia Panel (BioFire Diagnostics, Salt Lake City, UT) enables simultaneous detection of multiple respiratory pathogens as well as AMR genes directly from respiratory specimens within approximately one hour [4,6]. Previous multicenter studies have demonstrated that the FilmArray Pneumonia Panel provides higher sensitivity than conventional culture while substantially shortening the time to pathogen identification [4,6,7].

Beyond improving diagnostic accuracy, rapid molecular testing has the potential to influence downstream clinical management by enabling earlier microbiology-guided antibiotic decisions and supporting antimicrobial stewardship interventions [8]. Delayed pathogen identification contributes to prolonged empirical broad-spectrum antibiotic use, which is associated with adverse drug events, antimicrobial resistance, prolonged hospitalization, and mortality [9]. Although several studies have shown that rapid molecular diagnostics facilitate earlier antibiotic modification, their effects on overall antibiotic exposure, antibiotic de-escalation, and clinical outcomes have been inconsistent [5,8,10,11]. Consequently, real-world evidence regarding the clinical impact of multiplex molecular diagnostics remains limited.

Despite these advantages, concerns remain regarding the relatively lower specificity of molecular diagnostic panels because they may detect colonizing organisms or nonviable bacteria [6]. Therefore, molecular test results should be interpreted in conjunction with clinical, microbiological, and radiologic findings. Furthermore, most previous studies have focused primarily on intensive care unit (ICU) populations [12–14] or endotracheal aspirate specimens [13,15], whereas relatively few real-world studies have directly compared bronchoalveolar lavage (BAL) and sputum specimens using multiplex molecular assays [7,16].

This study evaluated the diagnostic performance and clinical utility of the BioFire FilmArray Pneumonia Panel using both BAL and sputum specimens from hospitalized patients with pneumonia. Specifically, we evaluated pathogen distribution, antimicrobial resistance gene detection, polymicrobial infection patterns, and diagnostic performance compared with conventional culture. We also assessed the clinical impact of FilmArray implementation by comparing result-driven antibiotic actions, diagnostic turnaround time, the time from specimen sampling and microbiological result reporting to antibiotic action, total antibiotic days, hospital length of stay, and 30-day mortality between the pre-implementation and FilmArray periods.

Materials and methods

Results

Pathogen distribution detected by the FilmArray pneumonia panel

Among the 558 respiratory specimens analyzed using the FilmArray Pneumonia Panel, 359 (64.3%) were positive for at least one pathogen, whereas 199 (35.7%) were negative (Table 1).

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Table 1. Pathogens detected by the FilmArray Pneumonia Panel in bronchoalveolar lavage (BAL) and sputum specimens.

https://doi.org/10.1371/journal.pone.0358986.t001

Among the positive specimens, 595 bacterial pathogens, 10 atypical bacterial pathogens, and 110 viral pathogens were identified. The most frequently detected bacterial pathogen was Klebsiella pneumoniae (18.7%), followed by Pseudomonas aeruginosa (15.3%), Staphylococcus aureus (14.3%), and Haemophilus influenzae (12.3%). L. pneumophila was the most frequently detected atypical bacterial pathogen (60.0%). Among viral pathogens, parainfluenza virus (24.5%), influenza A virus (20.0%), and human rhinovirus/enterovirus (17.3%) were the most frequently detected. Viral pathogens were identified more often in sputum specimens than in BAL specimens.

Comparison of culture and FilmArray results

Among the 297 BAL specimens, 35.0% were positive by both conventional culture and the FilmArray Pneumonia Panel, whereas 41.1% were negative by both methods. The remaining specimens showed discordant results: 20.2% were culture-negative but FilmArray-positive, whereas only 3.7% were culture-positive but FilmArray-negative.

Similarly, among the 261 sputum specimens, 31.0% were positive by both methods and 40.2% were negative by both conventional culture and the FilmArray Pneumonia Panel. In contrast, 28.0% of sputum specimens were culture-negative but FilmArray-positive, whereas only 0.8% were culture-positive but FilmArray-negative.

At the organism-level, the FilmArray Pneumonia Panel detected substantially more bacterial pathogens than conventional culture in culture-negative BAL and sputum specimens, whereas only a limited number of bacterial pathogens were detected exclusively by conventional culture (Tables 2 and 3).

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Table 2. Bacterial pathogens detected exclusively by FilmArray Pneumonia Panel or by conventional culture in bronchoalveolar lavage specimens.

https://doi.org/10.1371/journal.pone.0358986.t002

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Table 3. Bacterial pathogens detected exclusively by FilmArray Pneumonia Panel or by conventional culture in sputum specimens.

https://doi.org/10.1371/journal.pone.0358986.t003

Antimicrobial resistance gene detection

A total of 212 AMR genes were detected, comprising 118 in BAL specimens and 94 in sputum specimens (Table 4). The most frequently detected AMR gene was CTX-M, accounting for 47.2% of all detected AMR genes, followed by mecA/C with MREJ (25.0%) and KPC (16.5%). The detected carbapenemase genes included NDM (6.1%), VIM (2.8%), IMP (1.9%), and OXA-48-like (0.5%). Overall, carbapenemase genes accounted for 27.8% of all detected AMR genes.

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Table 4. Antimicrobial resistance determinants detected by the FilmArray Pneumonia Panel.

https://doi.org/10.1371/journal.pone.0358986.t004

Baseline characteristics of the clinical-impact cohort

Baseline characteristics of the patients included in the clinical-impact analysis are summarized in Table 5. A total of 494 patients were included: 294 in the pre-implementation period and 200 in the FilmArray period. Baseline imbalances were observed for age, CRP, erythrocyte sedimentation rate, D-dimer level, and residence type, whereas the remaining baseline characteristics were generally comparable between the two groups. Accordingly, the primary multivariable analyses were adjusted for the three variables with complete data and standardized mean differences of ≥0.10 (age, CRP, and residence type), together with sex, ICU admission, and ECOG performance status, which were prespecified a priori because of their established prognostic importance in pneumonia. Erythrocyte sedimentation rate, procalcitonin, and D-dimer also had standardized mean differences of ≥0.10 but contained 6–8% missing data, predominantly in the pre-implementation period; therefore, these variables were evaluated in sensitivity analyses rather than included in the primary models. The same covariates were used for both propensity score weighting and multivariable adjustment to ensure consistency across the primary analyses.

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Table 5. Baseline characteristics of patients included in the clinical-impact analysis.

https://doi.org/10.1371/journal.pone.0358986.t005

Clinical impact of FilmArray testing

The primary clinical-impact endpoint was result-driven antibiotic action (initiation or modification). After adjustment for prespecified covariates, patients in the FilmArray period were significantly more likely to undergo antibiotic initiation or modification within five days after microbiological reporting than those in the pre-implementation period (40.5% vs. 26.5%; adjusted odds ratio, 1.86; 95% confidence interval [CI], 1.24–2.79; P = 0.003) (Table 6). The median turnaround time from specimen sampling to reporting of the final microbiological result was 3 days (interquartile range [IQR], 2–4) in the pre-implementation period and 0 days (IQR, 0–0) in the FilmArray period (mean difference, −3.17 days; 95% CI, −3.31 to −3.03; P < 0.001). Accordingly, the interval from sampling to the result-driven antibiotic action was more than three days shorter in the FilmArray period (3.91 ± 1.73 vs. 0.47 ± 1.23 days; adjusted mean difference, −3.53 days; 95% CI, −4.08 to −2.99; P < 0.001), whereas the interval from result reporting to the antibiotic action was shorter by approximately half a day (adjusted mean difference, −0.59 days; 95% CI, −1.07 to −0.10; P = 0.019), indicating that most of the gain derived from the faster diagnostic turnaround rather than from a faster clinician response once the result was available. The FilmArray period had a shorter hospital length of stay (adjusted mean difference, −1.61 days; 95% CI, −3.07 to −0.15; P = 0.031). However, the adjusted duration of antibiotic therapy during hospitalization was longer (adjusted mean difference, 1.86 days; 95% CI, 0.29–3.44; P = 0.021). No significant difference in 30-day mortality was observed between the two groups.

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Table 6. Clinical impact of the FilmArray Pneumonia Panel on antibiotic management and clinical outcomes.

https://doi.org/10.1371/journal.pone.0358986.t006

Patterns of antibiotic initiation and modification

When the whole clinical-impact cohort was taken as the denominator, result-guided initiation of antibiotics was markedly more frequent in the FilmArray period than in the pre-implementation period (27.5% vs. 3.4%; adjusted odds ratio, 9.20; 95% CI, 4.48–18.90; P < 0.001), and the proportion of patients in whom no antibiotic action followed the microbiological result fell from 73.5% to 59.5% (adjusted odds ratio, 0.54; 95% CI, 0.36–0.81; P = 0.003) (Table 7). Escalation (15.0% vs. 9.0%; P = 0.053) and de-escalation (8.2% vs. 4.0%; P = 0.092) of an ongoing regimen were numerically less frequent in the FilmArray period, but neither difference was statistically significant before or after adjustment. The overall four-category distribution differed between the periods (chi-square P < 0.001). Implementation of the panel therefore changed the character of result-driven decisions − towards initiation of targeted therapy in patients not yet receiving antibiotics − rather than the frequency of escalation or de-escalation. Nevertheless, even in the FilmArray period, 59.5% of patients had no antibiotic action attributable to the microbiological result within five days.

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Table 7. Antibiotic actions attributable to the microbiological result in the whole clinical-impact cohort.

https://doi.org/10.1371/journal.pone.0358986.t007

Discussion

In this real-world before-and-after observational study, the BioFire FilmArray Pneumonia Panel demonstrated high diagnostic sensitivity and broad pathogen coverage in both BAL and sputum specimens and was associated with clinically meaningful improvements in microbiology-guided antibiotic management. Whereas previous studies have primarily focused on the analytical validation or diagnostic performance of the FilmArray Pneumonia Panel, our study evaluated both diagnostic performance and downstream clinical impact in hospitalized patients with pneumonia, thereby providing a more comprehensive assessment of its clinical utility in routine clinical practice.

Consistent with previous international studies, the FilmArray Pneumonia Panel frequently detected clinically important bacterial pathogens, including K. pneumoniae, P. aeruginosa, and S. aureus [4,6]. These organisms are commonly associated with multidrug resistance and poor clinical outcomes. In addition, respiratory viruses and atypical bacterial pathogens were frequently identified, particularly in sputum specimens, highlighting the broad etiologic spectrum of lower respiratory tract infections and the limitations of conventional culture in detecting these pathogens.

A prominent finding of our study was the high proportion of specimens that were culture-negative but FilmArray-positive, particularly among sputum specimens. This observation reflects the well-recognized limitations of conventional culture, including reduced sensitivity following prior antibiotic exposure and its inability to detect fastidious or slow-growing organisms [2,6]. Similar findings have been reported in critically ill patients and patients with COVID-19, in whom early empirical antibiotic therapy substantially decreases culture yield [6,7,15]. Furthermore, because conventional culture does not detect respiratory viruses or atypical bacterial pathogens, part of this discordance is expected and should not necessarily be interpreted as false-positive FilmArray results.

Despite these advantages, conventional culture remains an indispensable component of the microbiological evaluation of pneumonia. Unlike targeted multiplex PCR assays, culture can detect a broader range of bacterial and fungal pathogens, including unexpected organisms not covered by the FilmArray panel. In addition, semi-quantitative culture results may help distinguish colonization from true infection, while culture enables phenotypic antimicrobial susceptibility testing and further molecular characterization. Accordingly, the FilmArray Pneumonia Panel should be regarded as a complementary diagnostic tool rather than a replacement for conventional microbiological culture.

Although previous systematic reviews have reported higher diagnostic odds ratios and better clinical agreement for BAL specimens than for sputum specimens [16,17], our study was not designed to compare diagnostic performance between specimen types. Furthermore, previous studies have reported only modest agreement between FilmArray results and the final clinical diagnosis of pneumonia [18], emphasizing that molecular findings should always be interpreted within the overall clinical context.

The detection of antimicrobial resistance (AMR) genes further underscores the clinical value of FilmArray testing. In our cohort, CTX-M and mecA/C with MREJ were the most frequently detected AMR genes, reflecting the high prevalence of extended-spectrum β-lactamase-producing Enterobacterales and methicillin-resistant S. aureus among hospitalized patients with pneumonia. During the FilmArray study period (2023), local antimicrobial resistance surveillance demonstrated that approximately 20% of E. coli and 30% of K. pneumoniae isolates were ESBL producers, whereas carbapenem resistance was identified in approximately 1% of E. coli and 10% of K. pneumoniae isolates. In addition, approximately 50% of S. aureus isolates were methicillin resistant. These regional resistance patterns highlight the importance of rapid identification of both ESBL- and carbapenemase-associated resistance determinants, as well as methicillin-resistant S. aureus, to support early optimization of antimicrobial therapy. These findings are consistent with previous antimicrobial stewardship studies demonstrating the importance of rapid resistance gene identification for guiding appropriate antimicrobial therapy [11,15,18]. Early identification of resistance determinants may facilitate timely escalation when resistant pathogens are present while avoiding unnecessary broad-spectrum therapy when resistance genes are absent [2].

Detection of multiple pathogens was common in our study, with more than half of FilmArray-positive specimens containing two or more detected microorganisms. This finding highlights the complex microbiology of pneumonia and illustrates an important advantage of multiplex molecular diagnostics over conventional culture and single-target molecular assays [11,16]. Recognition of multiple-pathogen detection may influence antimicrobial selection and overall treatment strategies; however, the clinical significance of each detected organism requires careful interpretation together with the patient’s clinical presentation and other microbiological findings [4,17]. As reported previously, multiple-pathogen detection was more frequent in sputum specimens than in BAL specimens [4]. Because no independent clinical adjudication was performed, the clinical significance of multiple-pathogen detection could not be determined, and these findings should not be interpreted as definitive evidence of true polymicrobial infection.

Despite these diagnostic advantages, the moderate specificity observed for the FilmArray Pneumonia Panel warrants careful interpretation. This limitation is largely attributable to its high analytical sensitivity, which may detect colonizing organisms, residual nucleic acids from nonviable bacteria, or microorganisms below the threshold of clinical significance. Consequently, positive molecular results do not necessarily indicate active infection and, if interpreted without adequate clinical correlation, may lead to unnecessary initiation or escalation of antimicrobial therapy. Therefore, FilmArray results should always be interpreted together with the patient’s clinical presentation, radiologic findings, and conventional microbiological results.

The relatively high cost of multiplex molecular testing may also limit its widespread implementation in routine clinical practice. Future studies evaluating the cost-effectiveness and scalability of the FilmArray Pneumonia Panel in different healthcare settings are warranted.

Beyond diagnostic performance, implementation of the FilmArray Pneumonia Panel facilitated earlier and more frequent microbiology-guided antibiotic management. Measured from specimen sampling, patients in the FilmArray period underwent a result-driven antibiotic action more than three days earlier than those in the pre-implementation period, whereas the gain measured from result reporting was only about half a day. Almost the whole of this benefit therefore derived from the faster diagnostic turnaround of the panel (median, 0 vs. 3 days) rather than from a faster clinician response once the result was available, supporting the role of rapid molecular diagnostics in improving the timeliness of antimicrobial decision-making, one of the principal goals of antimicrobial stewardship programs.

Previous studies have reported reductions in overall antibiotic exposure following implementation of the FilmArray Pneumonia Panel [19]. However, our adjusted analyses did not demonstrate this benefit. Instead, total antibiotic exposure during hospitalization was modestly longer in the FilmArray period despite earlier microbiology-guided antibiotic modification. One possible explanation is that rapid identification of clinically important pathogens and AMR genes prompted continuation or escalation of appropriate therapy in patients with microbiologically confirmed or more severe infections rather than premature discontinuation of antibiotics. These findings suggest that rapid molecular diagnostics primarily improve the appropriateness and timeliness of antibiotic management rather than simply reducing antibiotic use.

When the whole cohort was taken as the denominator, escalation and de-escalation of an ongoing regimen were numerically less frequent in the FilmArray period, but neither difference was statistically significant; what changed was the character of the decision, with a nine-fold increase in the adjusted odds of result-guided initiation of targeted therapy and a fall in the proportion of patients in whom the result changed nothing (73.5% to 59.5%). Nevertheless, the microbiological result still did not alter management within five days in 59.5% of patients in the FilmArray period, and we did not observe an increase in antibiotic de-escalation. Clinicians appeared to continue relying primarily on conventional culture and phenotypic antimicrobial susceptibility testing when making definitive antimicrobial decisions, consistent with previous reports [12,20,21]. This may reflect uncertainty regarding the interpretation of multiple-pathogen detection, the clinical significance of molecular findings, or the limited predictive value of resistance gene detection alone [12]. These findings emphasize that successful implementation of rapid molecular diagnostics requires close integration with antimicrobial stewardship programs as well as clinician education to optimize interpretation and clinical decision-making [3,20,22].

FilmArray implementation was also associated with a modest reduction in hospital length of stay after adjustment for baseline characteristics, although no significant difference in 30-day mortality was observed. Similar findings have been reported in previous stewardship-focused investigations evaluating multiplex molecular diagnostics [19,23]. Although causal relationships cannot be established because of the observational study design, earlier pathogen identification and more timely microbiology-guided antibiotic management may contribute to improved clinical efficiency and healthcare resource utilization.

Several limitations should be acknowledged. First, this was a retrospective before-and-after observational study; therefore, temporal changes in clinical practice, antimicrobial stewardship activities, patient case mix, as well as other unmeasured factors may have contributed to the observed differences between the two study periods. Although multivariable adjustment and propensity score weighting were performed to reduce baseline imbalances, residual confounding cannot be excluded. Second, conventional culture was used as the reference standard despite its limited sensitivity, which may have affected estimates of the apparent diagnostic specificity of the FilmArray Pneumonia Panel. Third, antimicrobial management was not standardized and may have been influenced by individual clinician judgment and institutional antimicrobial stewardship practices. Fourth, we did not evaluate the semi-quantitative bacterial load categories or cycle threshold values reported by the FilmArray Pneumonia Panel, nor did we systematically compare detected antimicrobial resistance genes with the results of conventional antimicrobial susceptibility testing. These parameters should be evaluated in future studies. Fifth, hospital stays exceeding 30 days were excluded in different proportions in the two study periods (31.6% vs. 20.5%). However, analysis of the full cohort without this restriction showed a larger effect in the same direction, suggesting that the primary estimate may have been conservative. Sixth, no routine reference method for respiratory viruses was available during the study period; therefore, the diagnostic performance of the viral targets could not be evaluated, and we were unable to determine how positive viral results influenced antimicrobial prescribing decisions. Finally, although the FilmArray Pneumonia Panel provides rapid microbiological information, antibiotic prescribing decisions are influenced by multiple clinical factors in addition to microbiological test results. Clinical improvement or deterioration, radiologic findings, inflammatory markers, and other patient-specific considerations may all contribute to treatment modification. Therefore, although antibiotic actions were evaluated only when they occurred within five days of result reporting, this temporal association should not be interpreted as establishing a causal relationship between FilmArray implementation and changes in antibiotic prescribing. Future prospective studies should evaluate the proportion of clinically actionable FilmArray results and their direct effects on antimicrobial stewardship and patient outcomes.

Conclusion

In conclusion, the BioFire FilmArray Pneumonia Panel demonstrated high diagnostic sensitivity and broad pathogen coverage in both BAL and sputum specimens. Compared with conventional culture, the panel detected additional microorganisms and antimicrobial resistance genes; however, this study was not designed to determine whether these additional detections represented the true causative pathogens or clinically relevant resistance mechanisms. Therefore, FilmArray results should always be interpreted in conjunction with conventional microbiological testing and the overall clinical context.

In the clinical-impact analysis, implementation of the FilmArray Pneumonia Panel facilitated earlier and more frequent microbiology-guided antibiotic management and was associated with a modest reduction in hospital length of stay. However, implementation was associated with modestly increased overall antibiotic exposure and did not significantly increase antibiotic de-escalation. These findings support the role of rapid multiplex molecular diagnostics as an adjunct to conventional microbiological testing for improving the timeliness and precision of antimicrobial therapy. Nevertheless, their full clinical benefit is likely to be realized only when integrated with structured antimicrobial stewardship programs.

Supporting information

Acknowledgments

The authors thank Myeongjin Seo, medical technologist, for the collection of laboratory test results, and Seung-Hee Shin, a health information manager, for medical record review working at Gyeongsang National University Changwon Hospital..