Lower respiratory tract infections (LRTIs) continue to be a significant global health problem, despite advances in medical treatments and antibiotic therapies. The 2021 global statistics reflect the staggering burden of LRTIs, with approximately 344 million cases reported worldwide. These infections account for over 2.18 million deaths annually, with Streptococcus pneumoniae responsible for 505,000 of these deaths [1]. LRTIs is not only contribute to high mortality rates, but they also place a tremendous strain on healthcare systems, especially in low- and middle-income countries where access to advanced medical interventions is limited. The impact of LRTIs is particularly severe among children under five years of age, with 502,000 deaths occurring in this demographic annually. Although there has been significant progress in reducing the overall mortality rate from LRTIs, they remain a serious public health issue, especially in countries with limited healthcare resources [2]. One of the most pressing challenges in treating LRTIs is the rising prevalence of multidrug-resistant (MDR) organisms, such as Pseudomonas aeruginosa and Acinetobacter baumannii, which complicate the use of standard antibiotic therapies [3,4]. These bacteria, particularly in hospital settings, are often responsible for severe cases of VAP and other nosocomial infections [5]. The increasing resistance to conventional antibiotics has significantly reduced the effectiveness of intravenous (IV) treatments, creating a need for alternative therapeutic approaches [6].

In this context, nebulized antibiotics offer a promising solution by delivering high concentrations of drugs directly to the site of infection in the lungs, overcoming the limitations of systemic antibiotic delivery [7]. However, the lungs present numerous challenges for antibiotic penetration when drugs are administered systemically [8]. Blood-air barriers, poor perfusion in infected areas, and the thick mucus often present in LRTIs impede the ability of IV antibiotics to reach effective concentrations in the infected lung tissue. This is especially problematic in critically ill patients with conditions such as VAP, where traditional IV antibiotics often fail to adequately clear infections. Nebulized antibiotics bypass these physiological barriers by directly delivering the drug to the respiratory tract [9]. This localized administration not only improves drug concentration at the site of infection but also reduces systemic exposure, thereby minimizing the risk of side effects such as nephrotoxicity, which is common with high doses of systemic aminoglycosides. For example, nebulized formulations of tobramycin and colistin have been shown to achieve significantly higher local concentrations in the lungs compared to their IV counterparts [10]. This makes nebulized antibiotics particularly effective against MDR organisms in the lungs, as these pathogens typically require higher local concentrations of antibiotics for successful eradication.

Given the increasing antibiotic resistance and the complexity of treating respiratory infections in critically ill patients, the use of nebulized antibiotics has become an area of growing interest in the treatment of LRTIs. This review explores the current evidence, efficacy, and limitations of nebulized antibiotic therapies in the treatment of LRTIs, particularly focusing on infections caused by Gram-negative and MDR pathogens. Through an in-depth analysis of clinical trials, pharmacokinetic studies, and safety profiles, this review study aims to provide a comprehensive understanding of the potential role of nebulized antibiotics in managing LRTIs in both hospital and community settings.

This review study was conducted through a comprehensive and systematic approach, drawing from a wide array of scientific literature and clinical studies on the use of nebulized antibiotics for the treatment of LRTIs, with a particular focus on infections caused by Gram-negative and MDR bacteria. The methodology was designed to ensure that the findings presented are evidence-based, thorough, and representative of the current state of knowledge in this field.

The first step in the research process involved an extensive literature search, using multiple academic databases such as PubMed, Cochrane Library, and Scopus. A carefully selected set of search terms was employed, including “nebulized antibiotics,” “lower respiratory tract infections,” “ventilator-associated pneumonia,” “Gram-negative infections,” and specific pathogen and antibiotic names like Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and other MDR Gram-negative pathogens, tobramycin, amikacin and colistin. The search was limited to peer-reviewed journal articles, clinical trials, meta-analyses, and systematic reviews published between 2000 and 2024 to capture the most up-to-date research on this subject.

Inclusion and exclusion criteria were established to ensure the relevance and quality of the studies selected. Articles were included if they focused on the efficacy, safety, or pharmacokinetics of nebulized antibiotics in adult patients with LRTIs, particularly those involving Gram-negative or MDR bacteria. Studies that provided data on bacterial eradication rates, clinical outcomes, and adverse effects were prioritized. Exclusion criteria were applied to studies that focused solely on pediatric populations or non-respiratory bacterial infections, as well as research that was not available in English. This process ensured that the final pool of studies was highly relevant to the research questions addressed in the review.

Once the relevant literature had been identified, each study underwent a detailed review. Particular attention was given to randomized controlled trials (RCTs) and large-scale meta-analyses, as these provide the most robust evidence regarding the efficacy of nebulized antibiotics. Data were extracted from these studies to compare the outcomes of nebulized antibiotic therapies with those of conventional intravenous treatments. Key parameters, such as bacterial clearance rates, clinical response, mortality, and adverse events, were analyzed and synthesized to identify trends and conclusions.

Pathophysiology of LRTIs and the Role of Antibiotics

LRTIs encompass a broad spectrum of infections that affect the lungs and lower airways, including pneumonia, bronchitis, and bronchiolitis. Pneumonia, the most severe manifestation, is an infection of the alveoli, often caused by bacteria such as Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, and Gram-negative organisms like Pseudomonas aeruginosa and Acinetobacter baumannii. Viral pathogens such as respiratory syncytial virus and influenza also contribute significantly to LRTIs, particularly in pediatric populations and the elderly [11]. The immune response to these infections involves the recruitment of immune cells, inflammation of lung tissues, and the production of mucus, which can exacerbate airway obstruction and hinder effective gas exchange. This pathophysiology underscores the need for therapies that can penetrate lung tissues and clear infections while minimizing collateral damage to lung function [11].

Challenges in antibiotic penetration The anatomy and physiology of the lungs pose several challenges to systemic antibiotic delivery. The alveolar-capillary membrane, mucus production, and inflammatory processes during infections act as barriers to the effective penetration of drugs. Systemically administered antibiotics, such as β-lactams and aminoglycosides, often fail to achieve adequate concentrations at the site of infection in the lungs, leading to suboptimal treatment outcomes [9,12]. For example, aminoglycosides like tobramycin exhibit poor penetration into lung tissues when administered intravenously (IV), despite their efficacy in treating Gram-negative bacterial infections. The blood-air barrier and mucus can further limit the movement of these antibiotics from the bloodstream to the extracellular lining fluid (ELF) of the lungs, where bacteria are often located. This limited penetration is particularly problematic in the context of MDR organisms, which require higher local concentrations of antibiotics for eradication [13].

The efficacy of nebulized antibiotics is closely tied to factors such as the anatomy of the airways, patient ventilation, and aerosol particle characteristics. Optimal particle size plays a critical role in ensuring effective drug deposition in the lungs. Particles with a mass median aerodynamic diameter of 1–5 μm achieve over 90% deposition in the peripheral, alveolar-rich regions of the lungs. Particles smaller than 1 μm are often exhaled, while those larger than 5 μm tend to deposit in the oropharynx or within the device [14]. An ideal nebulized antimicrobial agent possesses properties such as lipophilicity, a positive charge, and high molecular weight, which help maintain high drug concentrations in the lungs while minimizing systemic absorption. This targeted drug delivery maximizes the treatment efficacy for pulmonary infections, especially those caused by MDR organisms.

History of inhalation therapy The history of inhalation therapy for respiratory infections dates back thousands of years. Ancient practices included figures like Pythia at Delphi inhaling vapors and Claudius Galen recommending sulfur inhalation. Over time, the concept evolved, with Pedanius Dioscorides in the 1st century prescribing nebulized sulfur. In 1932, the term “aerosol” was introduced, and advancements in nebulization devices during both World Wars improved treatment outcomes. In the 1940s and 1950s, nebulized antibiotics like neomycin, polymyxin, and penicillin G were tested for pneumonia, though warnings about risks arose by 1975. Modern developments include the use of nebulized tobramycin for cystic fibrosis and Pseudomonas infections in the late 1990s and 2000s. However, data on nebulized antibiotics for non-cystic fibrosis patients remain limited.

Advantages of nebulized antibiotics Nebulized antibiotics offer a targeted therapeutic approach that bypasses many of the limitations of systemic therapy. By delivering high concentrations of the drug directly to the lungs, nebulized antibiotics enhance local drug levels at the site of infection, potentially leading to better bacterial eradication and clinical outcomes [15]. This localized delivery minimizes systemic exposure, reducing the risk of nephrotoxicity and other side effects associated with high-dose IV antibiotic use.

Moreover, nebulized antibiotics can reach areas of the lungs that are difficult to access with systemic treatments. In patients with VAP, for example, bacteria often colonize the lower respiratory tract, where systemic antibiotics may not reach in sufficient concentrations. Nebulized therapies can overcome this barrier, delivering the drug directly to the infected alveoli and bronchioles [16].

Pharmacokinetic considerations The pharmacokinetics (PK) of nebulized antibiotics differ significantly from those of systemic antibiotics. Nebulized drugs are absorbed through the alveolar epithelium into the ELF, where they can achieve much higher concentrations than IV-administered drugs. For instance, studies have shown that nebulized tobramycin can reach ELF concentrations that are 100-fold higher than those achieved with IV administration [17]. This high local concentration is critical for treating infections caused by MDR organisms, which often require concentrations several times higher than the minimum inhibitory concentration (MIC) to be effective [18]. However, the PK of nebulized antibiotics can vary based on factors such as particle size, nebulizer efficiency, and patient-specific characteristics like lung function. In critically ill patients, mechanical ventilation and mucus production can further influence drug deposition, necessitating careful consideration of dosing and delivery methods [19].

Nebulized device Nebulizers are devices that deliver medication directly to the lungs in the form of aerosolized mist. The Table 1 summarized the difference between 3 types of nebulizers [16,20].

Table 1 . Summary of the features and differences between Jet, Ultrasonic, and Mesh nebulizers.

Device type	Jet nebulizer	Ultrasonic nebulizer	Mesh nebulizer
Mechanism	Uses compressed air to create an aerosol mist	Uses high-frequency vibrations to generate mist	Liquid is pushed through a vibrating mesh to create mist
Advantages	Cheap, easy to use Effective for drugs not suitable for pMDI and DPI	Easier to use, more efficient than Jet Shorter nebulization time Adjustable droplet size (<5 µm)	Quiet operation, portable Shorter treatment time Higher efficiency than other types Small droplet size (<5 µm)
Disadvantages	Low efficiency, difficult to clean Large droplet size (>5 µm) Requires compressed air and tubing	Large volume Cannot nebulize thick solutions Higher temperature (10–14°C) Large device size	Expensive, difficult to clean Cannot nebulize viscous solutions or drugs that crystallize when dry

Current Nebulized Antibiotics: Efficacy and Limitations

Availability of nebulized antibiotics The Table 2 presents the availability and approval status of various nebulized antibiotics used primarily for managing infections in cystic fibrosis patients. It highlights that while some antibiotics, such as nebulized tobramycin and amikacin, are available in the U.S., others, like nebulized aztreonam and powdered tobramycin, are not. In Vietnam, nebulized tobramycin is accessible, but the dry powder formulation and several others remain unavailable. Notably, colistimethate in dry powder form is available in Vietnam but not in the U.S., reflecting regional differences in antibiotic availability. These discrepancies underscore the importance of regulatory approval processes and market access, which can significantly influence treatment options for patients with respiratory infections.

Table 2 . Availability and approval status of nebulized antibiotics.

Nebulized antibiotic	Approved indication	Approving agency	Status in the U.S.	Status in Vietnam
Aztreonam	Improve respiratory symptoms in patients aged ≥7 years with Pseudomonas aeruginosa	FDA	Available	Not available
Tobramycin	Manage cystic fibrosis patients aged ≥6 years with Pseudomonas aeruginosa	FDA	Available	Available (Topi nebuliser)
Powdered tobramycin	Manage cystic fibrosis patients aged ≥6 years with Pseudomonas aeruginosa	FDA	Available	Not available
Amikacin (ALIS [Arikayce®])	MAC-LD	FDA	Available	Not available
Dry powder colistimethate for aerosolization	Manage chronic pulmonary infections due to Pseudomonas aeruginosa in cystic fibrosis patients aged ≥6 years	European Medicines Agency	Not available	Available (Colistin TZF)

MAC-LD=mycobacterium avium complex lung disease, FDA=U.S. Food and Drug Administration..

Nebulized tobramycin Tobramycin is one of the most commonly used nebulized antibiotics, particularly for treating chronic Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). Tobramycin inhalation solutions have been shown to improve lung function, reduce bacterial load, and decrease the frequency of exacerbations in CF patients [21]. The success of nebulized tobramycin in CF has prompted investigations into its use for non-CF patients with LRTIs, including those with VAP and bronchiectasis [22].

However, the evidence for nebulized tobramycin in non-CF patients is mixed. While some studies suggest that nebulized tobramycin can improve clinical outcomes in patients with VAP caused by Pseudomonas aeruginosa, others have found no significant benefit over systemic antibiotics alone. For example, a randomized controlled trial comparing nebulized tobramycin with placebo in VAP patients found no significant differences in clinical response or mortality [23].

Nebulized amikacin Amikacin, another aminoglycoside, has shown promise as an nebulized therapy for Gram-negative infections, particularly in patients with MDR pathogens. A recent clinical trial evaluating nebulized amikacin in VAP patients reported improved bacterial clearance and clinical response rates compared to IV therapy alone. The study found that patients receiving nebulized amikacin had a 1.51 times higher likelihood of bacterial eradication (p<0.0001) and a 1.23 times higher clinical response rate (p<0.0001) [24].

Despite these promising results, nebulized amikacin did not significantly impact mortality rates, ICU stay duration, or mechanical ventilation days. This highlights one of the key limitations of nebulized antibiotic therapy: while it may improve local bacterial clearance, its impact on overall clinical outcomes remains uncertain [25,26]. Further studies are needed to determine the long-term benefits of nebulized amikacin, particularly in combination with systemic antibiotics.

Nebulized colistin Colistin is frequently used as a last-resort antibiotic for treating infections caused by MDR Gram-negative organisms like Pseudomonas aeruginosa and Acinetobacter baumannii. The administration of nebulized antibiotics should be limited to patients with VAP caused by extensively drug-resistant (XDR) Gram-negative bacteria that are only sensitive to colistin or aminoglycosides. Several meta-analyses have demonstrated that in such infections, combining nebulized colistin with intravenous (IV) colistin results in improved patient outcomes compared to IV colistin alone [27-30]. However, it remains unclear whether nebulized antibiotics can reduce the development of bacterial resistance, as indicated by studies conducted in patients with ventilator-associated tracheobronchitis [31]. Furthermore, nebulized colistin has been associated with adverse effects such as bronchospasm and airway irritation, limiting its use in some patients [32].

Combination inhalation therapies In addition to monotherapy, there is growing interest in combination inhalation therapies, particularly for treating MDR infections. Combining nebulized antibiotics with mucolytics or anti-inflammatory agents may enhance drug delivery and reduce airway resistance, improving clinical outcomes. For example, studies have explored the combination of nebulized tobramycin with dornase alfa, a mucolytic agent used to reduce mucus viscosity in CF patients [33].

Similarly, combination therapies involving nebulized colistin and systemic antibiotics like rifampicin have been investigated for treating Acinetobacter baumannii infections. Preliminary data suggest that such combinations may enhance bacterial clearance and reduce the risk of resistance development, although further clinical trials are needed to confirm these findings [34].

Safety Considerations for Nebulized Antibiotics

Bronchospasm and airway irritation One of the primary concerns with nebulized antibiotic therapy is the risk of bronchospasm and airway irritation. This is particularly relevant for antibiotics like colistin, which can cause bronchoconstriction in some patients. In a study involving critically ill patients with VAP, 12% of those receiving nebulized colistin experienced an increase in serum creatinine levels, indicating potential nephrotoxicity, while 8% experienced bronchospasm [35]. The risk of bronchospasm can be mitigated by using pre-treatment bronchodilators and optimizing the formulation of nebulized antibiotics to reduce airway irritation [36]. For example, the nebulized formulation of aztreonam was specifically designed to minimize airway irritation by excluding arginine, which is present in the IV formulation and has been shown to cause lung function decline when nebulized [37].

Systemic absorption and nephrotoxicity Although nebulized antibiotics are primarily delivered to the lungs, systemic absorption can still occur, particularly with drugs like aminoglycosides that are known for their nephrotoxic potential. Nebulized amikacin and tobramycin have been associated with elevated serum levels in some patients, raising concerns about potential kidney damage, especially in patients with pre-existing renal impairment [38,39]. To minimize the risk of nephrotoxicity, careful monitoring of serum drug levels is recommended, particularly in patients receiving prolonged nebulized antibiotic therapy [40]. Additionally, the use of vibrating mesh nebulizers, which produce smaller and more uniform aerosol particles, may help reduce systemic absorption and improve drug delivery to the lungs [39].

Using intravenous (IV) antibiotics for aerosolization Using IV antibiotics for aerosol administration presents several concerns. First, antibiotics designed for inhalation and IV use are not interchangeable, and using non-nebulized formulations can lead to serious side effects, including death [32]. Nebulized antibiotics are specifically optimized to ensure proper drug delivery to the lungs, with suitable particle size and consistent dosing. Additionally, these formulations are designed for better tolerability; for example, IV aztreonam contains arginine, which can impair lung function when nebulized, so the nebulized version is arginine-free [41]. Furthermore, preservatives in IV antibiotics may irritate the airways if used in aerosol form.

Clinical Guidelines and Evidence from Randomized Controlled Trials

IDSA 2024 guidelines The 2024 guidelines from the Infectious Diseases Society of America (IDSA) provide specific recommendations regarding the use of nebulized antibiotics in clinical practice. Notably, the IDSA does not recommend the routine use of nebulized antibiotics for treating MDR Gram-negative infections, including those caused by Pseudomonas aeruginosa and Acinetobacter baumannii, in patients with VAP [42].

The 2024 IDSA guidelines on the use of nebulized antibiotics for treating pneumonia caused by DTR P. aeruginosa and CRAB highlight several important considerations. First, technical factors, such as aerosol particle size, nebulizer type, carrier gas properties, and respiratory settings, significantly affect drug delivery and deposition in the lungs, but these aspects may not be adequately emphasized in clinical trials. Recent studies suggest that using vibrating mesh nebulizers or low-flow ultrasound can enhance drug distribution, while high humidity and temperature can increase particle size, thus reducing efficacy [43]. Second, the optimization of aerosol delivery methods is crucial, as trials using IV formulations instead of specialized aerosol formulations may not achieve maximum effectiveness. Studies indicate that although drug concentrations in pulmonary epithelial lining fluid are high, nebulized antibiotics may not penetrate lung tissue adequately for significant antibacterial activity [15]. Third, there is a lack of consistency in trial design and clinical criteria, which can lead to heterogeneous results, making it essential to conduct larger, more carefully designed trials to confirm the efficacy of nebulized antibiotics.

Key randomized controlled trials Several key RCTs have investigated the efficacy of nebulized antibiotics in treating LRTIs, particularly in critically ill patients with VAP. One of the largest trials, which included INHALE trial evaluated the efficacy of nebulized amikacin, used alongside standard intravenous antibiotics, as an adjunctive treatment for Gram-negative pneumonia in mechanically ventilated patients [25]. Conducted across 153 intensive care units in 25 countries, the double-blind, placebo-controlled, phase 3 study involved 725 randomized patients. Both groups received either 400 mg amikacin or placebo nebulized every 12 hours for 10 days, in addition to intravenous antibiotics. The primary endpoint, survival at days 28–32, showed no significant difference between the groups, with 75% survival in the amikacin group and 77% in the placebo group. The study concluded that nebulized amikacin did not improve outcomes in these patients. Secondly, the IASIS trial investigated the safety and efficacy of the amikacin fosfomycin inhalation system (AFIS) as an adjunctive therapy for VAP caused by Gram-negative bacteria. Conducted between 2013 and 2016, this randomized, double-blind, placebo-controlled phase 2 study involved 143 patients, comparing the effects of AFIS plus standard care with a placebo [26]. The primary outcome, the change in Clinical Pulmonary Infection Score (CPIS), showed no significant difference between the groups. Secondary outcomes, including mortality rates and ventilator-free days, were also not significantly different. Although the AFIS group had fewer positive tracheal cultures, the system did not improve overall clinical outcomes. The 3rd RCT investigated the safety and effectiveness of nebulized colistimethate sodium (CMS) as adjunctive therapy for VAP caused by Gram-negative bacteria, including multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, at Siriraj Hospital in Thailand. The study involved 100 patients, with one group receiving nebulized CMS and the other receiving nebulized saline alongside systemic antibiotics. While the CMS group showed a significantly better microbiological outcome (60.9% vs. 38.2%), there was no significant difference in clinical outcomes between the groups. The study concluded that nebulized CMS is safe, but its clinical benefits remain uncertain [44]. Similarly, a meta-analysis of 13 RCTs involving 1,733 patients with VAP found that nebulized antibiotics may reduce bacterial loads and improve clinical symptoms, but they did not significantly affect survival or the need for mechanical ventilation. These findings underscore the need for more robust evidence to support the routine use of nebulized antibiotics in critically ill patients [24].

The lack of clinical benefit from nebulized antibiotics in trials remains unclear. While PK/PD modeling studies show that nebulized colistin can achieve high drug levels in the lung’s epithelial lining fluid, it is likely that nebulized antibiotics fail to penetrate lung tissue adequately, limiting their bactericidal effect. This is partly due to the use of parenteral formulations not designed for inhalation and suboptimal nebulizers like jet nebulizers. Professional opinions on the use of nebulized antibiotics as adjunctive therapy are divided. Due to inconsistent clinical benefits, uneven drug distribution, and risks of respiratory complications such as bronchoconstriction, nebulized antibiotics are not recommended for treating pneumonia caused by drug-resistant Pseudomonas aeruginosa or CRAB.

Current practices and knowledge gaps A recent survey on the use of nebulized medications in mechanical ventilation reveals significant insights into current practices and knowledge gaps among healthcare professionals. The study, which involved 854 ICU physicians across Europe, found that while 99% utilize aerosol therapy in ventilated patients, only 30% administer nebulized antibiotics to more than five patients per year. The most commonly used antibiotics include colistin (59%) and tobramycin (31%). However, practices appear suboptimal, with 77% of respondents not adjusting ventilator settings during aerosol delivery, 22% not ceasing humidification, and 33% failing to change the expiratory filters. Furthermore, knowledge regarding optimal droplet size and nebulizer performance was limited, as most participants could not provide correct answers [45]. Supporting data from another study involving 2,808 patients across 81 ICUs in 22 countries indicated that 24% received nebulized medications, primarily antibiotics, with 62% using jet nebulizers, 29% ultrasonic nebulizers, and 9% vibrating mesh nebulizers. Only 30% adjusted ventilator settings when administering nebulized antibiotics. In summary, despite high interest in nebulized antibiotics, there is a low implementation rate and insufficient knowledge regarding their management [46]. Therefore, guidelines from professional associations are necessary to enhance the use of nebulized antibiotics in mechanical ventilation.

Future Directions: Prevent VAP

The future prospects of nebulized antibiotics, such as amikacin, to prevent VAP show promise based on recent research [47]. In a multicenter, double-blind, randomized controlled trial, critically ill patients who had been on mechanical ventilation for at least 72 hours received nebulized amikacin or placebo for 3 days. The study found that 15% of patients in the amikacin group developed VAP compared to 22% in the placebo group, demonstrating a significant reduction in VAP incidence. Despite some trial-related adverse effects, the results suggest that short-term nebulized amikacin therapy could potentially lower the risk of VAP, signaling a positive direction for its future use in critical care settings [48].

In conclusion, the implementation of nebulized antibiotics requires meticulous attention to formulation, processes, and equipment, along with appropriate management strategies. The use of intravenous antibiotics for aerosolization poses significant risks and is not recommended. Nebulized antibiotics may offer advantages by achieving high drug concentrations at infection sites, potentially enhancing clinical and microbiological responses. However, RCTs have not demonstrated significant improvements in survival rates, duration of mechanical ventilation, or length of ICU stay. Furthermore, the IDSA 2024 guidelines do not endorse the use of nebulized antibiotics for treating resistant Gram-negative bacterial pneumonia, including MDR P. aeruginosa and Carbapenem-resistant Acinetobacter baumannii. Nonetheless, data from RCTs indicate that amikacin may be effective in preventing VAP in intubated patients, suggesting that further research is needed to clarify the role of nebulized antibiotics in this context.

This research was financially supported by Haiphong University of Medicine and Pharmacy.

We would like to thank all researchers whose work contributed to this review, as well as our colleagues for their invaluable support and guidance throughout this study.

No potential conflict of interest relevant to this article was reported.