This study aimed to investigate the influence of pulmonary surfactant on the antifungal activity of selected drugs; MCF, CAS, ANI, and AMB against C. krusei and C. albicans. Whereas considerable knowledge exists on the differential distribution of systemic antifungal agents in the lungs and their association with pulmonary alveolar macrophages and the epithelial lining fluid, little is known about the pharmacodynamic consequences and potential interactions of antifungal agents at these sites [11, 12]. Additive antifungal activity for example against Aspergillus fumigatus has been reported for the combination of amphotericin B formulations with pulmonary alveolar macrophages [13, 14], and that of echinocandins with monocyte-derived macrophages [15, 16]. In addition, in a set of comprehensive in vitro experiments, it was shown that posaconazole (and its parent itraconazole, but not voriconazole) concentrates within cell membranes of pulmonary epithelial cells and rapidly transfers to Aspergillus fumigatus, where it accumulates to high concentrations and persists at the site of its target enzyme to exert antifungal activity [17, 18].

The limited understanding of pulmonary surfactant's impact on antifungal drugs in invasive pulmonary mycoses has prompted exploration, given its critical role in preventing alveolar collapse and its potential as a vehicle for enhanced antibiotic drug delivery, as seen in previous research on daptomycin facing commercial challenges due to inhibited activity in its presence [5, 7, 8].

In the present study, surfactant demonstrated a variable impact on the antifungal activity of the tested drugs. CAS exhibited no significant change in activity, while AMB, MCF, and ANI exhibited a reduction in killing efficacy in the presence of surfactant, ranging from 1 to 5 log steps, against both C. krusei (e.g., ATCC 6258, displaying a 5 log step reduction at 1 × MIC of MCF with SUR present at 1 × 10^7 CFU/mL compared to without SUR at 1 × 10^2 CFU/mL) and C. albicans (e.g., ATCC 90028, showing a 4 log step reduction at 1 × MIC of ANI with SUR present at 1 × 10^7 CFU/mL compared to without SUR at 1 × 10^3 CFU/mL). The concentration-dependent inhibition of SUR observed in clinical isolates of C. krusei (as illustrated in Fig. 7) further underscores the nuanced interaction between surfactant and antifungal drugs, implying that the unique microenvironment of the alveoli could impact the therapeutic effectiveness of antifungal drugs.

In contrast to C. krusei, the data regarding the impact of surfactant on C. albicans appears to be strain and antifungal specific. As noted by Gil-Alonso et al., Candida spp. exhibit varying susceptibilities to echinocandins, even within the same species. Therefore, when antifungal treatment fails, it may be attributed to the fact that different isolates of the same species do not respond equally to antifungals [19].

While our study sheds light on the surfactant-mediated impact on antifungal activity, the specific mechanisms remain elusive. Further investigation is warranted to understand the intricate interactions between pulmonary surfactant and antifungal drugs at a molecular level, offering deeper insights into the observed effects. It's important to note that post-antifungal effects have not been tested specifically in our study, even though this aspect might be of interest, particularly for echinocandins.

As described in a recent publication, surfactants contain high amounts of phospholipids, which could potentially underlie the observed interaction with antimicrobials [6]. Moreover, the origin of surfactant might also influence antifungal activity. Surfactant formulations can vary in their composition, as phospholipids may be sourced from bovine or porcine origins, potentially impacting this interplay [6]. Investigating these factors could provide valuable insights into optimizing antifungal therapy in the context of pulmonary surfactant.

This study provides valuable insights into the variable impact of pulmonary surfactant on antifungal activity. Strengths include the comprehensive assessment of multiple antifungal drugs and the consideration of both standard strains and clinical isolates. In the guideline "Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America," emphasis is placed on the recognition that invasive infection caused by Candida spp. is primarily linked to medical progress, constituting a major contributor to morbidity and mortality in healthcare settings. While there are over 15 Candida spp. responsible for human disease, more than 90% of invasive cases are attributed to the 5 most prevalent pathogens: C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei. Despite their individual differences in virulence potential, antifungal susceptibility, and epidemiology, collectively, infections caused by these organisms are commonly referred to as invasive candidiasis [20]. Moreover, in Erami et al.'s study, colonization with Candida was identified in 69 out of 100 immunosuppressed COVID-19 patients, highlighting the prevalence of Candida spp. colonization [21]. These studies support our decision to choose C. albicans and C. krusei isoaltes as relevant test strains in our study.

Moreover, different surfactant concentrations (from 0.01 mg/L to 1 mg/L) have been tested to cover a range of physiological surfactant concentrations achieved in the patient’s lung. However, the limitations lie in the in vitro nature of the study, necessitating further validation in clinical settings, and the need for exploration of potential mechanisms (e.g. molecular investigations) underlying the observed effects. Furthermore, the use of porcine-derived surfactant, Curosurf®, may not entirely capture the characteristics of human surfactant. However, obtaining human surfactant involves invasive lavage procedures, presenting a limitation in the study.

In conclusion, this in vitro study sets the stage for further research to validate and extend these findings in clinical settings. Additionally, this study prompts consideration that assessing antifungals in standard growth media could lead to an overestimation of the agents' efficacy at the intended site of action. Future studies should explore the underlying mechanisms, assess the clinical relevance testing concentrations of antimycotics reached at the target sites, and consider the broader implications for the treatment of invasive pulmonary mycoses.