Chronic obstructive pulmonary disease (COPD) remains a major global public health challenge and is among the leading causes of morbidity and mortality worldwide (Boers et al. 2023; GOLD 2025). Acute exacerbations of COPD (AECOPD) are clinically significant events characterized by worsening respiratory symptoms requiring additional treatment and frequently resulting in hospitalization. These episodes contribute substantially to disease progression, impaired quality of life, increased healthcare utilization, recurrent hospital admissions, and mortality (Waeijen-Smit et al. 2024).

Respiratory infections are recognized as one of the principal triggers of AECOPD. Current evidence indicates that exacerbations may result from bacterial infections, viral infections, or mixed viral-bacterial etiologies, highlighting the complexity of pathogen identification in routine clinical practice (Yu and Yang 2025). Respiratory viral infections are responsible for approximately 50% of COPD exacerbations and are associated with more severe symptoms, secondary bacterial infections, synergistic deterioration of lung function, and prolonged hospitalization (Guo-Parke et al. 2020).

Despite the heterogeneous etiology of AECOPD, empirical antimicrobial therapy is frequently initiated at hospital admission because differentiation between bacterial and viral infections remains challenging. Although antibiotics are beneficial in selected patients with suspected bacterial infection, inappropriate or prolonged antimicrobial use may contribute to antimicrobial resistance and unnecessary antibiotic exposure. Gram-negative bacteria represent an important group of pathogens among hospitalized patients with AECOPD, with Pseudomonas aeruginosa among the commonly identified bacterial pathogens and substantial antimicrobial resistance reported among Gram-negative isolates (Yu and Yang 2025). Recent evidence has demonstrated that multiplex polymerase chain reaction (PCR) significantly improves pathogen detection and facilitates earlier optimization of antimicrobial therapy compared with conventional microbiological methods, supporting its integration into antimicrobial stewardship programs (Hua and Sethi 2024; Serapide et al. 2025).

Clinical pharmacists represent key members of multidisciplinary antimicrobial stewardship teams. Their responsibilities include interpretation of microbiological findings, evaluation of antimicrobial appropriateness, identification of drug-related problems, and support of antimicrobial escalation, de-escalation, or discontinuation decisions (Dighriri et al. 2023). In patients with AECOPD, pharmacist-led antimicrobial stewardship interventions have demonstrated significant improvements in antibiotic prescribing quality, supporting the expanding role of clinical pharmacists in optimizing antimicrobial therapy and promoting rational antibiotic use in routine clinical practice (Gong et al. 2022; Nguyen et al. 2026). Furthermore, the integration of rapid multiplex PCR diagnostics into pharmacist-led antimicrobial stewardship interventions may provide additional value by supporting antimicrobial escalation and de-escalation decisions and promoting more targeted antimicrobial use (Wang et al. 2025).

Despite growing evidence supporting the effectiveness of pharmacist-led antimicrobial stewardship interventions, evidence regarding the integration of rapid multiplex PCR diagnostics into the management of AECOPD remains limited. This gap is particularly relevant in low- and middle-income healthcare settings, where conventional microbiological culture remains the cornerstone of pathogen identification and where both antimicrobial stewardship programs and access to advanced molecular diagnostic technologies are still in the early stages of development. Therefore, assessing the clinical utility of multiplex PCR-guided antimicrobial stewardship may provide valuable evidence for optimizing antimicrobial prescribing and supporting early therapeutic decision-making.

The aim of this study was to evaluate the clinical utility of multiplex PCR diagnostics integrated into a clinical pharmacist-led antimicrobial stewardship model in hospitalized patients with AECOPD. Specifically, the study assessed the contribution of pharmacist-interpreted multiplex PCR testing to respiratory pathogen detection, pharmacist-guided antimicrobial optimization, and clinical outcomes.

A total of 70 patients hospitalized due to AECOPD were included in the study. The study population was predominantly male, comprising 46 patients (65.7%).

Baseline laboratory assessments showed abnormal inflammatory and biochemical parameters in a substantial proportion of the study population. The median leukocyte count was 10.91 × 10⁹/L (IQR 8.91–13.74), while the median CRP concentration was 16.05 mg/L (IQR 8.64–51.58). Elevated LDH levels were also observed, with a median value of 325 U/L (IQR 264.75–477.25). The median blood glucose concentration at admission was 6.89 mmol/L (IQR 5.87–9.43). Overall, elevated CRP levels were present in 60 patients (85.7%), followed by elevated blood glucose in 52 patients (74.3%), elevated LDH levels in 46 patients (65.7%), and leukocytosis in 40 patients (57.1%).

Pulmonary function testing revealed severe airflow limitation within the study population. The median FEV₁ was 32.0% of the predicted value (IQR 26.0–43.75), while the median FVC was 49.0% of the predicted value (IQR 38.5–60.5). The median FEV₁/FVC ratio was 53.39% (IQR 44.0–62.79), consistent with persistent airflow obstruction characteristic of COPD. Bronchitis and emphysema were present in 38 patients (54.3%) and 32 patients (45.7%), respectively.

Respiratory pathogens were identified in 45 patients (64.3%). Because multiple pathogens were detected in several patients, the cumulative number of pathogen detections exceeded the total number of patients with positive microbiological findings. Regarding the distribution of identified microorganisms, influenza A virus and respiratory syncytial virus (RSV) were the most frequently detected pathogens, each identified in 10 patients (14.3%). These were followed by Pseudomonas aeruginosa and human rhinovirus/enterovirus, detected in nine patients each (12.9%), and Streptococcus pneumoniae in eight patients (11.4%). Human coronavirus OC43 (HCoV-OC43), Candida spp., and human metapneumovirus were identified in six patients each (8.6%), while Haemophilus influenzae and Escherichia coli were detected in five patients (7.1%) and four patients (5.7%), respectively. Less frequently detected pathogens included influenza B virus and Moraxella catarrhalis (two patients each, 2.9%), whereas Trichosporon spp., Klebsiella pneumoniae, and Serratia marcescens were each identified in one patient (1.4%). Analysis of infection patterns in the overall study population demonstrated a predominance of viral pathogens and mixed infections. Single viral infections were identified in 11 patients (15.7%), while single bacterial infections were detected in nine patients (12.9%). Multiple viral infections involving two or more viral pathogens were observed in three patients (4.3%), whereas multiple bacterial infections were identified in two patients (2.9%). Viral-bacterial co-infections represented the most frequent mixed infection pattern and were detected in 14 patients (20.0%). Viral-fungal co-infections were less common, occurring in three patients (4.3%). Fungal infections were identified in two patients (2.9%), while multiple fungal infections were detected in one patient (1.4%).

While multiplex PCR enabled the detection of respiratory viral pathogens that are not routinely identified by conventional microbiological culture methods, it also increased the detection of several bacterial pathogens. Conventional microbiological culture remained important for the identification of fungal pathogens, including Candida spp. and Trichosporon spp. (Table 1). Multiplex PCR additionally detected Serratia marcescens together with the VIM (Verona integron-encoded metallo-β-lactamase) resistance marker in one patient, providing early information on the presence of a clinically relevant antimicrobial resistance mechanism.

Intravenous empirical antimicrobial therapy was initiated in all patients at hospital admission. With respect to empirical antimicrobial treatment, moxifloxacin represented the predominant therapeutic choice and was administered in 35 patients (50.0%). Ceftriaxone and azithromycin were the next most frequently used agents, prescribed in 16 patients (22.9%) and 10 patients (14.3%), respectively. In contrast, ceftazidime, meropenem, and piperacillin/tazobactam were used less frequently, accounting for three (4.3%), four (5.7%), and two (2.9%) prescriptions, respectively.

Following multiplex PCR testing, pharmacist-guided antibiotic de-escalation was performed in 44 patients, while empirical antimicrobial therapy was maintained in 17 patients. Additional targeted antimicrobial optimization was implemented in selected cases according to the identified pathogen profile (Tables 2, 3).

The median length of hospitalization was 12 days (range 5–25). At discharge, laboratory findings demonstrated improvement in inflammatory and biochemical parameters. The median CRP concentration decreased to 3.0 mg/L (IQR 3.0–5.6), accompanied by a reduction in the median leukocyte count to 9.36 × 10⁹/L (IQR 7.25–12.3). Median blood glucose and LDH levels decreased to 6.23 mmol/L (IQR 5.22–8.26) and 300 U/L (IQR 200–388), respectively.

Point-biserial correlation analysis was performed to evaluate the association between microbiological, clinical, and laboratory variables and hospital length of stay. In the unadjusted analysis, human rhinovirus/enterovirus infection (r = 0.41, p = 0.0003) and elevated LDH at admission (r = 0.29, p = 0.013) were positively associated with hospital length of stay. After Bonferroni correction for multiple comparisons, only the association between human rhinovirus/enterovirus infection and hospital length of stay remained statistically significant (adjusted p = 0.010), whereas the association with elevated LDH was no longer statistically significant (adjusted p = 0.436). No other variables showed statistically significant associations after Bonferroni correction (Table 4).

Human rhinovirus/enterovirus infection, which remained statistically significant after Bonferroni correction, and elevated LDH at admission, based on its clinical relevance and significant association in the unadjusted analysis, were further evaluated using linear regression models. In the univariable analysis, human rhinovirus/enterovirus infection (β = 5.74, 95% CI: 2.71–8.77, p = 0.0003) and elevated LDH (β = 2.85, 95% CI: 0.60–5.10, p = 0.0136) were each associated with longer hospital length of stay. In the multivariable model, both human rhinovirus/enterovirus infection (β = 5.39, 95% CI: 2.45–8.34, p = 0.0005) and elevated LDH (β = 2.48, 95% CI: 0.40–4.56, p = 0.020) remained associated with longer hospital length of stay. The multivariable model explained 23.8% of the variability in hospital length of stay (R² = 0.238; adjusted R² = 0.215). Variance inflation factors were approximately 1.01 for both variables, indicating no evidence of multicollinearity.

Infrequently detected pathogens, including Klebsiella pneumoniae, Serratia marcescens, and Trichosporon spp., were reported descriptively only because their low frequency precluded reliable inferential statistical analysis.

Because residual diagnostics indicated some departure from normality, the association between human rhinovirus/enterovirus infection and hospital length of stay was additionally evaluated using the Mann–Whitney U test. Consistent with the regression analysis, patients with human rhinovirus/enterovirus infection had a significantly longer hospital stay than those without this infection (median 20 [IQR 14–21] vs. 11 [IQR 9–14] days; U = 433, p = 0.005). Hospital length of stay was also compared according to different infection patterns using the Mann–Whitney U test. No statistically significant differences were observed between patients with and without viral, bacterial, fungal, or mixed infections (all p > 0.05). Descriptively, patients with multiple viral infections had a longer median hospital stay than those without multiple viral infections (20 vs. 11 days; p = 0.106), and fungal infections were associated with a longer median hospitalization (18 vs. 11.5 days; p = 0.115). Patients with viral-bacterial co-infections also had a slightly longer median hospital stay (12.5 vs. 11 days; p = 0.392). In contrast, bacterial infections, multiple bacterial infections, and viral-fungal co-infections were associated with shorter median hospital stays than the corresponding comparison groups (all p > 0.05).

The findings of the present study support the potential role of clinical pharmacist-led antimicrobial stewardship in optimizing empirical antimicrobial therapy among hospitalized patients with AECOPD. In the present setting, the clinical pharmacist was directly involved in the management of the molecular diagnostics laboratory and interpretation of multiplex PCR results for respiratory pathogen detection, enabling timely integration of microbiological findings into clinical decision-making and antimicrobial stewardship interventions. Following the availability of multiplex PCR results, antibiotic de-escalation was performed in the majority of patients, with therapeutic modifications typically implemented within the first 24 h after the results became available.

A major finding of the present study was the substantially higher diagnostic yield of multiplex PCR compared with conventional microbiological culture. Most stewardship interventions were associated with viral pathogens detected exclusively by multiplex PCR. In total, 43 viral pathogen detections were identified across six viral categories. Because some patients had more than one viral pathogen detected, this number refers to pathogen detections rather than individual patients. These findings are consistent with a previous study demonstrating that rapid molecular diagnostics significantly improve respiratory pathogen detection and provide clinically actionable results considerably earlier than conventional microbiological methods, thereby facilitating timely antimicrobial optimization (Echavarría et al. 2018). However, despite their superior diagnostic performance, the clinical utility of multiplex PCR testing in the management of acute exacerbations of chronic obstructive pulmonary disease remains less well established than in pneumonia, as many currently available assays were originally developed and validated for pneumonia diagnosis (Hua and Sethi 2024). In this context, the present study provides real-world observational evidence suggesting that the integration of multiplex PCR diagnostics into a clinical pharmacist-led antimicrobial stewardship framework may support early pathogen identification and targeted therapeutic decision-making. Furthermore, the majority of antimicrobial stewardship interventions in the present study were triggered by viral pathogens detected exclusively by multiplex PCR, suggesting the potential clinical value of routine viral testing in a patient population in which microbiological evaluation has traditionally focused predominantly on bacterial pathogens.

After adjustment for multiple comparisons, human rhinovirus/enterovirus was the only microbiological variable that remained significantly associated with hospital length of stay. In the univariable regression model, patients positive for human rhinovirus/enterovirus had an estimated 5.74-day longer hospital stay, while the association remained similar after adjustment for elevated LDH in the multivariable model. The corresponding model explained only a modest proportion of the observed variability in hospital length of stay, indicating that additional clinical and patient-level factors not captured in the present study are likely to contribute to hospitalization duration. Nevertheless, only nine patients were positive for human rhinovirus/enterovirus, and most detections occurred in the context of co-infection. Five patients had viral-bacterial co-detections involving human rhinovirus/enterovirus and one or more bacterial pathogens (Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, or Haemophilus influenzae), with respiratory syncytial virus additionally detected in two of these patients. Two additional patients had multiple viral detections involving human rhinovirus/enterovirus with respiratory syncytial virus or human coronavirus OC43, while only two patients had human rhinovirus/enterovirus detected as a single pathogen. Therefore, the observed association should be interpreted with caution and confirmed in larger studies before attributing an independent effect of human rhinovirus/enterovirus on hospital length of stay. Notably, the longest hospitalization observed in the study population (25 days) occurred in a patient with combined respiratory syncytial virus, human rhinovirus/enterovirus, and Escherichia coli infection. These findings suggest that the observed association between human rhinovirus/enterovirus detection and prolonged hospitalization may not be attributable solely to isolated rhinovirus/enterovirus infection but may also reflect its frequent occurrence in mixed viral and viral-bacterial infections, which are generally associated with greater clinical complexity and disease severity. Previous studies have identified human rhinovirus as one of the most common viral triggers of acute exacerbations of COPD and have demonstrated its ability to enhance airway inflammation, impair antibacterial host defenses, and facilitate secondary bacterial infection (Love and Proud 2022). Furthermore, rhinovirus-associated exacerbations have been linked to delayed recovery, increased symptom burden, and greater healthcare utilization (Ritchie and Wedzicha 2020). The present findings are consistent with these observations and further highlight the potential clinical value of rapid human rhinovirus/enterovirus detection, particularly in patients with co-infections, where early identification may support more appropriate antimicrobial management and help avoid unnecessary antibacterial exposure.

A further strength of the present study was the integration of a clinical pharmacist-led molecular diagnostics laboratory into the antimicrobial stewardship workflow. This approach extends previous evidence demonstrating that pharmacist-led antimicrobial stewardship interventions improve the appropriateness of antibiotic prescribing, reduce unnecessary antimicrobial exposure, and promote guideline-concordant therapy among hospitalized patients with acute exacerbations of chronic obstructive pulmonary disease (Iovino 2023; Nguyen et al. 2026). The findings of the present study are consistent with recent evidence demonstrating that multiplex PCR testing not only improves pathogen detection but also facilitates early recognition of clinically relevant antimicrobial resistance mechanisms, thereby supporting timely antimicrobial optimization (Serapide et al. 2025). In the present study, multiplex PCR additionally detected Serratia marcescens together with the VIM carbapenemase resistance marker in a clinical specimen, providing early information on the presence of a potentially clinically relevant resistance mechanism and thereby supporting timely antimicrobial stewardship decision-making. This observation highlights the potential value of rapid molecular diagnostics in informing antimicrobial therapy beyond pathogen identification alone.

This study has several limitations. First, its observational single-arm design, without a comparator group receiving antimicrobial stewardship without multiplex PCR guidance, does not allow conclusions about the independent contribution of multiplex PCR to clinical outcomes. Second, the study was conducted at a single tertiary-care center with a relatively small sample size, which may limit the generalizability of the findings. In addition, several pathogens were detected in only a small number of patients, limiting pathogen-specific analyses. Although multiple statistical comparisons were performed, the association between human rhinovirus/enterovirus and hospital length of stay remained statistically significant after adjustment for multiple testing. However, this finding was based on a small number of rhinovirus/enterovirus-positive patients and should be confirmed in larger prospective studies. The study also included only in-hospital outcomes and did not include long-term follow-up. Therefore, mortality, hospital readmissions, and other longer-term outcomes were not evaluated.

This observational study suggests that integrating multiplex PCR diagnostics into a clinical pharmacist-led antimicrobial stewardship program may support antimicrobial optimization in hospitalized patients with AECOPD. Clinical pharmacists played an important role in interpreting multiplex PCR results and translating microbiological findings into patient-specific antimicrobial stewardship recommendations. This approach may facilitate timely therapeutic decision-making and more appropriate antimicrobial use in routine clinical practice.