Candida species is able to result in clinically invasive infections, commonly referred to as invasive candidiasis (IC). It can lead to skin and mucosal lesions, fungemia, and occasionally multiple focal infections. The pattern of its symptom varies with the different infectious sites (1). The infection caused by Candida spp. is among the top three infections commonly taking place in the intensive care units (ICUs) worldwide (2, 3), accounting for 18% of all infections (4). In particular, annually there are hundreds of thousands of patients who are inflicted with IC globally. Owing to its high mortality (about 70%), IC has been regarded as an emerging threat to public health (2). Since patients from the ICUs usually harbor impaired physiological and immune functions, probably because of the underlying diseases, clinically invasive interventions, hormone treatments, or hospitalization duration (5), they are prone to developing IC. The different Candida spp. differ in the severity of invasive infections, treatment strategies, and disease prognosis. As the widespread utilization of broad-spectrum antifungal agents and prophylactic empirical treatments are extensively applicable in clinic, the IC infection rate among the acute and severe patients is annually elevated. The alteration of fungal community composition and antimicrobial resistance has posed a potential threat to the clinical diagnosis and treatment of IC (6, 7). Timely and rational usage of antifungals is of great significance to the prognosis of those patients in the ICUs. Hence, it is critically essential for better early treatment and improvement of clinical outcomes of the patients to investigate the epidemiological features and to profile the antifungal sensitivity of IC in local regions.

In the present study with the associated clinical data, we evaluated the species distribution and antifungal agent sensitivities of Candida spp., and the clinical features and the diverse risk factors of ICU patients with IC. Moreover, the 14-α-sterol demethylase (ERG11) and sterol δ5,6-desaturase (ERG3) genes of azole-resistant C. tropicalis were sequenced to explore the molecular mechanism of drug resistance. Meanwhile, the therapeutic effects observed from the patients with azole-resistant C. tropicalis were explicitly documented in current study.

IC is increasingly involved with hospital-acquired infections. It usually gives rise to relatively high morbidity and mortality, especially among the acute or severe patients in the ICUs. These ICU patients are often treated with clinically invasive interventions, broad-spectrum antibiotics and/or hormonotherapy. Consequently, the frequent invasive interventions will be able to compromise the mucocutaneous barrier protection. Extensive utilization of multiple antifungals will inevitably raise drug resistance. And hormonotherapy is prone to host immunosuppression, increasing the infection risk of IC (2). It is essential to early diagnose the disease and immediately give appropriate antifungals to treat ICU patients with IC (11). The exogenous source of hospital-borne infections in the ICUs may be person-to-person contact, contaminated equipment or building services in hospital (5). Therefore, reducing interpersonal cross-infections and microbial colonization of equipment/devices is pivotal to prevent candidiasis in ICU patients.

Since the coronavirus disease 2019 outbreak during the study period and the implementation of the hierarchical medical system in China (12) that classifies distinct degrees of diseases according to doctors' diagnosis, medical institutions at all levels provide continuing medical services, medical resources are allocated based on demands in recent years, more acute and severe patients such as critically ill patients with diabetes and (solid organ) transplantations have preferentially been admitted to the ICU in our Province-level tertiary hospital, seemingly resulting in a relatively high proportion of the infections. This may not be the case for the actual average infection rate. Therefore, no specific analysis was directly or arbitrarily conducted in the present study on the issue of the overall infection rates. Although C. albicans was earlier the dominant pathogen accounting for two-thirds of the infections, an increasing number of NCAC can currently be identified and responsible for almost 50% of the infections (13). Similarly, our data revealed that C. albicans was still the principal pathogen causing invasive fungal infections in our ICU patients, while C. glabrata and C. tropicalis were the two major NCAC of invasive candidiasis. Nevertheless, studies reported that C. parapsilosis was the most common NCAC for invasive candidiasis in ICU patients in other parts of China (14). Various risk factors, alone or in combination, influence the frequency and nature of Candida infections, including specimen source, age, length of hospital stay, underlying diseases, etc. (11, 15, 16). In our study, urine, BALF, and blood were the main sources of specimens. To be noted, the majority of Candida spp. were harvested from urine, which presented a different source pattern with other studies (17, 18). That difference is likely attributable to the seasonal, regional and environmental preferences of the fungi including genus Candida. Furthermore, the patient's underlying disease (e.g., diabetes) and immunosuppressive state (e.g., with renal transplantation) may primarily contribute to the relatively high incidence of Candida urinary tract infections in this study.

The patients with IC involved were nearly elderly and experienced relatively long length of hospital stay, more than half were over 50 years old and with >14 days of ICU stay. Amidst the indicated risk factors, tracheotomy, diabetes mellitus and concomitant bacterial infection were highly indicative of the occurrence of invasive candidiasis in ICU patients in the present study. Invasive procedure, e.g., tracheotomy, is regularly a key factor entailing an increased chance of Candida colonization/infection in ICU patients. Previous studies showed that diabetes mellitus was able to predispose one to systemic candidiasis due to several factors, among which the progression of microvascular disease remarkably contributed to the mechanism of lowered host defense, and the diabetic vasculopathy exacerbating hypoperfusion and hyperglycemia and likely leading to neutrophil and lymphocyte dysfunctions with impaired opsonization (19, 20). The clinical significance of polymicrobial interactions, particularly those between bacteria and fungi with high pathogenic potentials, remains largely underestimated, although several studies reported that bacterial infection might be a risk factor for disseminated candidiasis. It is necessary to raise its awareness in the treatment and management of immunocompromised individuals (21, 22). The specific mechanisms of interaction between bacterial and fungal infections require further investigations.

The antifungal agents can be divided into different categories, including azoles, polyenes, fluoropyrimidine analogs, echinocandins, morpholines, allylamines, thiocarbamates, and 5-FC (23). In this study, we analyzed the sensitivity of Candida spp. to five antifungal drugs commonly used in clinic. Our results demonstrated that all of the strains were sensitive to amphotericin B, and over 90% of them exhibited susceptibility to 5-FC (only 2.60% of C. albicans were resistant). Other researchers declare that the pathogenic spectrum of Candida infections has changed over the past decade, with a gradual shift from C. albicans to NCAC that may be less susceptible to azoles (24). The Candida spp. in this study presented a significant diversity in sensitivity to the individual members of azoles. Briefly, C. tropicalis showed relatively low sensitivity to fluconazole, voriconazole and itraconazole; C. albicans were all sensitive to all the members of azoles; C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei were comparatively less susceptible to itraconazole. Currently, itraconazole has only oral formulations available and is generally reserved for patients with mucosal candidiasis or outpatient use, nevertheless itraconazole is not well recommended to treat patients with IC which likely prefer its intravenous formulations if supported by large well-controlled clinical trials (25, 26). Therefore, the drug sensitivity test of itraconazole conducted in this study depicted its in vitro efficacy against Candida spp., which potentially provides knowledge for possible azole cross-resistance, concomitant Candida infections, and better antifungal drug designs in future. These observations produced similar drug sensitivity patterns with other studies, but with slight differences (27, 28); for instance, no fluconazole-resistant C. glabrata was observed in the present study, which probably is the current drug resistance feature of our local region, though this difference may also be caused by the limited sample size. Hence, our data indicate that there might be a species-dependent susceptibility of invasive fungi to azoles. Frequent use of fluconazole in the treatment of IC may be a cause of the increased antifungal resistance in ICU patients. These findings have enormous potential in guiding the use of antifungal drugs for ICU patients with IC.

The mechanisms of Candida resistance include altered drug affinity and/or target abundance, reduced drug uptake via efflux pumps, and formation of biofilms which resist drug action (29, 30). Certain C. tropicalis we found exhibited a high-level resistance to fluconazole, itraconazole, and voriconazole, and even presented a cross-resistance effect. C. tropicalis drug resistance mechanism is closely associated with gene mutations (mainly ERG11 and ERG3) (31). Our findings further unveiled double homozygous mutations of A395T and C461T in ERG11 gene among all azole-resistant C. tropicalis strains, resulting in the amino acid substitutions of Y132C and S154C in ERG11. These mutations are believed to attenuate the affinity of ERG11 for azoles and give rise to azole resistance (32, 33). Several studies reported in vivo synergistic effects of azole plus caspofungin on multidrug-resistant C. glabrata and C. albicans (34, 35). Notably, co-administration of posaconazole plus echinocandins was well tolerated without changing the pharmacokinetics of either agent (36). Similarly, in the present study, despite other factors that may affect the outcome of treatment, two of the three patients with deterioration were treated only with azole monotherapy, while the other four patients with controlled infection were treated with the combination of azole plus echinocandins antifungals. However, due to limited case size, whether the combination therapy could be an alternative to monotherapy for patients with azole-resistant C. tropicalis needs to be further addressed.

In summary, this study reported the Candida species distribution and their antifungal sensitivities, and clinical characteristics of ICU patients with invasive Candida infections in central part of China. Our results showed that the age, length of hospitalization, tracheotomy, diabetes mellitus as well as concomitant bacterial infection could be considered as the main risk factors for candidiasis. The polymorphism of the ERG11 gene in C. tropicalis may be closely associated with azole resistance.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, MZ703041; https://www.ncbi.nlm.nih.gov/, MZ703040.

The studies involving human participants were reviewed and approved by the Life Ethics Committee of Anhui Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

BW, YH, and JX conceived and designed the experiments. BW, JX, YW, and YL designed the research protocol and performed the experiments. BW, JX, and MZ performed data acquisition and analysis. BW, XH, FL, YL, MZ, YH, and JX contributed to the interpretation of results and assisted in writing the manuscript. All authors read and approved the final manuscript.

This study was supported by the National Natural Science Foundation of China (81601446) (BW) and the Natural Science Foundation of Anhui Province (1708085QH210) (BW). The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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