In this study, we designed a PN panel to evaluate the consistency between bacterial pathogens and AMR using routine culture methods. This is the first study to assess the significance of such a panel in clinical practice in China. Additionally, we analyzed the detection of the PN panel in cases of CAP and HAP. The bacterial panel and AMR both demonstrated a PPA of 85% compared with the conventional culture method. Apart from the advantages of point-of-care testing and rapid cultivation, the bacterial panel exhibited 25% more clinical benefits than the culture method. Also, early detection of AMR genes helps to adjust antibiotics in a timely manner, thereby avoiding complications and reducing the cost of antibiotics.

Bacteria were the most frequently detected pathogens in the PN panel, with A. baumannii and K. pneumoniae being the bacteria most strongly associated with HAP, in line with findings from previous studies [23, 24]. Unlike in a previous study, we found that S. aureus (14.62%) was more prevalent in patients with CAP than S. pneumoniae (7.69%), which might be related to the inclusion of nearly one-third of immunosuppressed patients in our study. As previously reported, S. aureus, P. aeruginosa, and K. pneumoniae are the most common bacterial infections in immunocompromised patients [25]. Hence, rapid bedside etiological testing is crucial as the etiology is linked to the patient’s underlying disease, disease severity, and even the site of onset. We observed that coinfection was more prevalent in patients with HAP than in those with CAP. Previous research has indicated that coinfection is significantly associated with disease severity and high mortality [26]. A study from Korea reported that 13.6% of patients with CAP had coinfection, while the proportion increased to 21.9% in patients with severe CAP [27]. The PN panel can simultaneously detect various bacteria, viruses, and even atypical pathogens, which may serve as an early warning indicator for the patient’s condition, whereas sputum bacterial cultures can only detect one pathogen at a time. In conclusion, the PN panel, as a multiplex detection reagent, may provide enhanced clinical practice guidance for physicians.

Consistent with the findings of a previous study [28], our study indicated that the PN panel detected more bacterial targets than the culture method, resulting in a 24.06% increase in patients reported as positive using the PN panel, with relatively high PPA (85%) and NPA (92%). Consequently, negative results may be employed for early antibiotic de-escalation, as the negative predictive value exceeded 90% in the PN panel. Similarly, prior research has reported superior performance of PN panels for bacterial detection, with PPAs ranging from 90.0 to 98.4% and NPAs ranging from 93.8 to 98.1% [21, 29,30,31]. While four K. pneumoniae strains were detected in the bacterial culture but not in the PN panel, resulting in a PPA of only 73%, Stenotrophomonas maltophilia, B. cepacia, and Providencia skrjabini were only detected using the culture method, whereas S. pneumoniae, H. influenzae, S. aureus, and P. aeruginosa were frequently missed.

Previous studies have demonstrated that quantitative PCR can distinguish symbiosis from pathogenicity by observing the charge [32], such as 103 CFU/mL used for the protected specimen brush or 104 CFU/mL of BAL used as an indicator to discontinue antibiotics against VAP [32]. The PN panel is semi-quantitative, with levels of 104, 105, 106, and 107 for bacterial targets, which is significant for guiding the initiation of antibiotic therapy in patients with HAP. In our study, an increasing number of semi-quantitative PN panels improved the likelihood of sputum cultures containing the same pathogen. The highest proportion of culture methods producing the same pathogen was observed when the PN panel detected bacterial targets of 107 copies/mL or greater. Conversely, the corresponding pathogen was not detected in the sputum culture when the bacterial target was 104 copies/mL, as detected using the PN panel. This suggests that it may be challenging to detect crucial organisms at extremely low concentrations, even though they are still associated with diseases. Further research is needed to explore whether the detection of unidentified, low-abundance, cultured microorganisms in the PN panel is of prognostic importance. Studies have also indicated that a high level of semi-quantitative signal intensity of positive microorganisms detected using multiple PCRs is closely related to positive bacterial cultures [29, 33], which may be useful for interpretation in the clinical applications of PN panels.

Molecular tests for genetic markers associated with antibiotic resistance, such as mecA, carbapenemases, and ESBLs, have been associated with positive outcomes, including reduced duration of optimal antibiotic therapy, shorter ICU stays, and decreased mortality rates [28, 34]. Our study demonstrated that hospitalization costs, antibiotic consumption, and the incidence of complications were higher in patients with drug-resistant genes than in those without drug-resistant genes. This emphasizes that early identification of drug resistance information and corresponding clinical interventions can help reduce economic costs and the occurrence of complications. Prior research has shown that the concordance rate for accessible resistance targets was 79% (14/18), consistent with phenotypic susceptibility testing [35], whereas in our study, the proportion of consistency in the phenotypic sensitivity test was 85% (52/61). Notably, mecA/C and MREJ of the PN panel exhibited extremely high predictive values for methicillin resistance, with 100% PPA and NPA in patients with positive S. aureus culture. Previous studies have indicated that the PPA for mecA/C and MREJ detection with PN panels was 100%, but NPA was < 90% [31]. However, further research is required to fully evaluate the PN panel, as our study included only four samples with positive S. aureus cultures.

We have previously described real-time PCR for the detection of NDM, KPC, VIM, IMP, and OXA-48, which are currently the most prevalent carbapenemase-producing genes [36]. In this study, the rate of phenotypic carbapenem resistance was relatively high, with 78.26% (36/46) of the specimens showing carbapenem resistance, the most common strains being A. baumannii and K. pneumoniae. Among the 30 carbapenem-resistant strains cultured, carbapenem-resistant genes were detected in 24 samples using the PN panels, while the remaining 6 were not detected. The six resistant strains were P. aeruginosa (three cases) and A. baumannii (three cases). This may be mediated by mechanisms other than carbapenem enzymes, such as the overexpression of efflux pumps or reduction of outer membrane pore proteins in Pseudomonas spp. [37]. Additionally, the overexpression of efflux pumps plays a significant role in the resistance of A. baumannii to tigecycline and imipenem [38]. Similarly, CTX-M testing demonstrated a positivity rate of 90%. However, 67% of these patients harbored concomitant carbapenemase genes. Considering that carbapenemase resistance often results in cephalosporin resistance [39, 40], the actual predictive efficacy of this measure may be diminished. These genetic tests facilitate the prompt addition of antibiotics and the implementation of appropriate isolation measures.

Our study has several limitations. First, we did not compare multiple specific etiological methods but rather culture results for bacteria with PN results. For example, bacterial culture is the primary method for the clinical diagnosis of S. pneumoniae; however, the detection rate of this method is relatively low and is influenced by various factors. Hence, urine antigen or other PCR tests should also be considered. Second, the methods of both analyses in this study were derived from the same alveolar lavage, but not the same specimen, which may have led to slight differences in the study results, although it is more in line with real-world research. Additionally, in this prospective study, we did not compare the clinical outcomes of the PN panel with those of standard methods. Our study revealed that patients in the HAP group had higher detection rates, a greater abundance of pathogens, and higher rates of resistance; however, the specific differentiation of clinical benefits was not achievable. Resistance genes influenced the clinical outcomes in our study, strongly supporting the necessity of detecting resistance genes in patients with LRTIs, though the cost of the panel will be higher than conventional culture. More prospective randomized studies are necessary to assess the impact of PN panels on the clinical outcomes of infected patients, including the types of pathogens and coinfections.