Within this combined clinical and experimental investigation, an NGS-based diagnostic approach and plasmatic measurements of MR-proADM and/or IL-17A were identified to be suitable for a comprehensive, reliable, and fast diagnosis of mycoses in patients suffering from septic shock. Moreover, ADM appeared to be one key element of various conserved host response patterns during epithelial infections with Candida spp.; however its functional characterization needs to be determined in further experimental studies.
3.1. Invasive Fungal Infections—A Clinical Dilemma
In patients suffering from severe sepsis or septic shock, positive blood cultures are obtained in only one third of cases despite a proven underlying bacterial infection [
31,
32,
33]. This is partially attributable to technical shortfalls in blood culture acquisition, but is also due to very low rates of viable microorganisms or fastidious organisms in the blood stream [
34]. Especially culture-based diagnostics for the detection of fungemia are known to be associated with relevant weaknesses [
35]. Although a fungal infection might be present in up to 30% of patients with severe sepsis or septic shock, only 2–3% of these infections can be proven by blood culture [
31]. Accordingly, a molecular approach with higher sensitivity for sepsis might, therefore, overcome the aforementioned limitations of classic microbiological diagnostics. However, state of the art molecular approaches based on PCR amplification of target sequences suffer from limited power to discriminate between contaminations, colonization, and infections, often revealing ambiguous or invalid results. Moreover, up to now there are still no commercial assays available for the identification of fungal isolates [
26]. The present clinical investigation therefore aimed to assess the incidence, risk factors and outcome relevance of fungal infections in patients with septic shock and also tried to evaluate the additional benefit of a next-generation sequencing (NGS)-based diagnostic procedure in affected patients.
According to the Candida-Score of Leon et al. [
36] (
Table S3) and in line with the results of Leroy et al. [
37], patients participating in the present clinical investigation were identified to be at high risk of a fungal infection. In line with the recent literature, fungal pathogens were found in our cohort in two thirds of all septic patients, whereas patients with fungal isolates were shown to be either colonized in 66.6% or suffered from an invasive infection in 33.3% [
14]. Resulting fungal isolates were also comparable to previous epidemiologic studies, in which
Candida spp. (
C. albicans and
C. glabatra) were isolated most frequently [
14,
31,
38,
39]. Moreover, the incidence of invasive pulmonary Aspergillosis (caused by
Aspergillus fumigates) was comparable to previously published investigations [
10,
14,
40,
41].
All patients suffered from septic shock and therefore need to be considered as critically ill, as assessed by various disease severity scores (e.g., APACHE II, SOFA and SAPS II). Although these scores did not differ significantly between patients with or without fungal isolates as previously described by Lichtenstern et al. [
14], the SOFA-score was shown to be significantly increased in patients suffering from a fungal infection in comparison to patients with a fungal colonization (
Table 2) as well as patients without a fungal infection (
Table 3). Accordingly, disease severity and the presence/occurrence of fungal infections seem to be closely related. The same holds true for the requirement of renal replacement therapy, mechanical ventilation as well as tracheostomy, which were all significantly increased in either patients with fungal isolates in comparison to those without fungal isolates (
Table 1), or in patients with a fungal infection in comparison to patients with a fungal colonization (
Table 2), respectively patients without a fungal infection (
Table 3) [
42,
43].
Within the presented investigation, the overall mortality was 22% and 34% at 28 and 90 days respectively. Although this mortality is substantially lower than the APACHE II predicted mortality of 55–75% [
44], the presented mortality data are in line with a previously published investigation of our workgroup [
14]. Moreover, mortality of patients suffering from a fungal infection within the presented investigation is in line with the current literature, reporting mortality rates of 30–70% due to aspergillosis and of 20–70% due to candidiasis [
14,
45,
46,
47,
48]. Surprisingly, the presence of fungal isolates, with respect to a fungal infection, did not result in a deterioration of the patient’s outcome (as assessed by the 28 or 90 day mortality) within the presented investigation as previously described by Montravers et al. [
49] or Lichtenstern et al. [
14], which might probably be attributable to the small cohort of patients included within the recent investigation. However, in line with the current literature, the length of ICU as well as hospital stay were significantly prolonged and the rate of fascia dehiscences was significantly increased in patients showing fungal isolates or suffering from a fungal infection [
49] in comparison to the different peer groups (
Table 1,
Table 2 and
Table 3). In line with the results of Fiore et al. and Theocharidou et al., special attention should also be placed on the role of the liver in the context of mycoses, since pre-existing liver cirrhosis as well as liver surgery prior to sepsis onset have been closely related to the occurrence of a fungal infection [
50,
51].
3.4. Fungal Biomarkers for the Diagnosis of Fungal Infections
Nowadays, especially the use of non-invasive diagnostic tools such as BG or GM is aimed at attenuating the above mentioned inefficiencies in the diagnosis of invasive fungal infections, although these biomarkers are also far from perfect. Several studies have addressed the potential usefulness of BG and GM for the diagnosis of an invasive fungal infection in neutropenic as well as non-neutropenic patients [
47,
58,
59,
60]. Accordingly, the potential value of both biomarkers has been acknowledged by their inclusion in the revised European Organisation for Research and Treatment of Cancer/National Institute of Allergy and Infectious Disease Mycoses Study Group (EORTC/MSG) criteria definitions of invasive fungal diseases/infections [
61].
BG is a common cell wall component of various medically important fungi, including
Candida spp. and
Aspergillus spp. Although plasmatic BG measurements can easily be performed by the use of commercially available assays, reports on the screening performance of BG are scarce, the test performance varies, and published data on BG are hallmarked by relevant limitations. Nevertheless, recent meta-analyses on the diagnostic value of plasmatic BG for the detection of patients with invasive fungal diseases suggest that the BG assay is a useful screening tool for the discrimination of patients with or without an invasive fungal infection [
62,
63,
64]. However, since the BG assay is not absolutely sensitive and specific for an invasive fungal infection, BG results should always be evaluated together with clinical and microbiological findings [
63,
64]. Accordingly, the workgroups of Digby et al. and Marty et al. were able to reveal false positive BG plasma levels in patients suffering from a bacterial infection [
65] or receiving an antibiotic treatment [
66]. These diagnostic deficiencies also became evident within the present investigation, since plasma levels of BG did not differ significantly between patients suffering from a fungal infection and colonized patients, or patients without any fungal isolates.
GM is a heat-stable polysaccharide present in the fungal wall of most
Aspergillus spp., a saprophytic filamentous fungus that can usually be isolated in the environment [
67]. In the course of an invasive disease, GM can be detected in different body fluids (e.g., serum, bronchoalveolar lavage fluid (BALF), cerebrospinal fluid). Most frequently, invasive Aspergillosis (IA) is an infection of the lower respiratory tract and primarily affects immunocompromised patients suffering from prolonged neutropenia or following hematopoietic organ transplants [
68]. However, IA has nowadays been recognized as an emerging infectious disease also in critically ill ICU patients in the absence of traditional IA risk factors (as described above, e.g. severe neutropenia >10 days, allogenic stem-cell transplant recipient, prolonged corticosteroids use, intake of T-cell immunosuppressants, inherited severe immunodeficiency) [
61,
69,
70,
71,
72]. Rather, chronic obstructive pulmonary disease (COPD), short-term corticosteroid use, acute respiratory distress syndrome (ARDS), severe sepsis/septic shock, H1N1 virus infection, decompensated chronic liver disease and acute renal failure are risk factors for IA in critically ill patients [
70,
71,
72]. Due to the aforementioned diagnostic weaknesses as well as a great heterogeneity in the different study populations, incidence and mortality rates of IA range from 0.02–19%, and from 46–95%, respectively [
70,
73]. Moreover, in some cases the proof of IA has only been made by post-mortem samplings of critically ill non-survivors, thus clearly underlying a diagnostic gap in the accuracy of recent diagnostic approaches for the detection of IA [
19,
20,
21]. Especially since a positive respiratory culture is present in only 40–50% of affected patients, non-invasive determination of GM in patients’ samples is known to be of value for the diagnosis of invasive Aspergillosis (IA) [
74]. Serum measurements of GM in high-risk neutropenic patients reveal a sensitivity as well as specificity of 82% and 81% respectively (cut-off 0.5 optical density index) [
75]. However, GM results in non-neutropenic patients are much worse due to a clearance of GM by neutrophils, thus limiting the suitability of GM serum levels for the diagnosis of IA in critically ill patients [
76]. Accordingly, either consecutive serum GM determinations [
77] or single measurements of GM in BALF [
78] were shown to provide a better diagnostic accuracy for the diagnosis of IA, whereas the optimal GM cut-off value in BALF (GM cut-off: 0.5 vs. 0.8 vs. 1.0) is still a matter of debate [
78,
79,
80]. Of note, also the combination of GM with BG is known to increase the specificity value for diagnosing IA. Thus, if clinical conditions are feasible, a combination of both assays is recommended in order to rule in diagnoses for at-risk patients [
63].
3.5. Immune Monitoring as a Diagnostic Tool in Fungal Infections
The human body is steadily confronted with a myriad of pathogens at its interfaces between inside and outside. Especially
Candida spp. frequently colonize mucosal surfaces throughout the body, but usually without inducing infection. However, this only works as our body has adapted to this condition by establishing physical barriers (e.g., mucosal and skin) as well as setting guardians to protect these barriers. Innate immunity represents the first line of defense against invading fungi, since neutrophils as well as monocytes/macrophages contribute to phagocytosis and direct killing of
Candida spp. In more detail, fungal pathogen associated molecular patterns (PAMPs, e.g., β-glucans or mannans) are mainly recognized by two types of pattern recognition receptors (PRRs), Toll-like receptor (TLR)-2 and TLR-4, or C-type leptin receptors (CLRs, mainly Dectin-1) on host cells, which are known to be upregulated in the case of an invasive fungal infection. These innate sensing mechanisms on dendritic cells (DCs), macrophages and epithelial cells ultimately lead to the activation of multiple intracellular pathways with the production of distinct sets of cytokines and other mediators, resulting in the differentiation of CD4
+ T helper (T
h) cells as well as regulatory T (T
reg) cells in response to fungi [
81].
A predominance of T
h1 cells arises from a TLR- as well as CLR-mediated activation of DCs throughout fungal PAMPs. Activation of T
h1 cells is associated with an enhanced synthesis of the signature cytokine IFN-γ, which is known to be essential for an optimal activation of phagocytes at the site of infection, thus promoting optimal fungal clearance. In contrast, failure of the IFN-γ dependent axis between T
h1 cells and phagocytes might be a risk factor for overwhelming fungal infections [
82]. Accordingly, patients suffering from a fungal infection within the presented investigation were characterized by significantly increased plasma levels of IFN-γ especially at later stages after sepsis onset (
Table S1).
Contrariwise, a predominance of T
h2 cells seems to counteract protective T
h1 cell responses due to an inhibition of fungal clearance, thus favoring fungal infections or a disease relapse [
83,
84,
85]. IL-4 was shown to be the signature cytokine for T
h2 cell depending inflammation; whilst in contrast an inhibition of IL-4 dependent pathways resulted in a restoration of antifungal properties [
86]. Within the presented investigation leveling of IL-4 was shown to be comparable to IFN-γ (
Table S1), thus suggesting an activation of both, T
h1 and T
h2 cell-dependent pathways in response to invasive fungal infections.
Apart from T
h1 and T
h2 cells, another T-helper cell type (named T
h17) has recently attracted attention in the context of fungal immunity [
87]: These cells are activated by IL-6, IL-23 as well as IL-1ß and are characterized by the ability to synthesize IL-17A, representing the signature cytokine of T
h17 cells. IL-17A is known to mobilize neutrophils, to activate phagocytosis of neutrophils and macrophages as well as to induce the synthesis of defensins by epithelial cells [
81,
87,
88,
89,
90]. Accordingly, IL-17 can be considered to be a key mediator of defense against candidiasis and its plasma levels might therefore be used for the diagnosis of patients suffering from an invasive fungal infection. Indeed, Krause and colleagues were recently able to show that candidemic patients have significantly higher IL-17A plasma levels in comparison to non-candidemic patients [
91], thus suggesting a potential role of IL-17A for the anticipation of invasive candidiasis. These results can be clearly supported by our investigation, since IL-17A was identified as a promising tool for the identification of patients suffering from a fungal infection. In parallel with significantly elevated plasma levels of IL-17A, IL-6 as the major activating cytokine of T
h17 cells also revealed significantly increased plasma levels in patients with a fungal colonization, respectively infection (
Table S1). However, one has to keep in mind that high IL-17A levels are especially associated with
Candida spp., whereas
Aspergillus spp. were shown to be poor inducers of IL-17A [
92]. Accordingly, the one patient suffering from IA within the presented investigation also revealed lower plasma levels at the time of diagnosis in comparison to patients suffering from an invasive Candidiasis. In summary, T
h17 cells seem to be of great importance for the host protection against fungi, and their signature cytokine IL-17A was identified to be a promising biomarker for the identification of patients suffering from an invasive candidiasis. Nevertheless, the release of this cytokine is not specific for fungal immune reactions. Elevated plasma levels of IL-17A may also occur in the context of bacterial sepsis, as described for a cecal ligation and puncture (CLP)-induced polymicrobial sepsis in mice [
93]. This may have led to an increase in IL-17A plasma levels even in study patients without a fungal infection, resulting in a relevant overlap between the different subgroups as well as leading to a relevant number of outliers in all subgroups of patients, especially in the early phase after sepsis onset.
In order to keep the balance between an effective elimination of the fungal invader and acceptable collateral tissue damage, another T-helper cell subtype was shown to be of relevance. Via their signature cytokine IL-10, so called regulatory T-cells (T
reg) are able to dampen the immune response, thus reducing damage to the host but conversely resulting in a state of immunosuppression [
81]. This might be due to the fact that IL-10 is known to be an inductor of T
h2 cells, thus counteracting the pro-inflammatory effects of an IFN-γ driven T
h1 cell response in fungal immunity. Accordingly, an inverse relationship between IL-10 and IFN-γ was suggested by several investigators [
82] that may potentially result in a high susceptibility to fungal infections especially in those patients presenting with high IL-10 levels [
94]. However, within the presented investigation of our workgroup, high IL-10 levels could be observed in patients suffering from a fungal colonization or infection in parallel with elevated IFN-γ levels, thus being a consequence, rather than a cause of the underlying fungal infection.
Adrenomedullin (ADM) represents a vasodilatory peptide hormone of 52 amino acids, which is released in several disease states such as heart failure, renal failure, respiratory failure, liver cirrhosis, cancer, as well as infectious diseases incl. sepsis [
95]. The synthesis of ADM is widely distributed in several tissues throughout the whole body (kidney, lung, heart, blood vessels, etc.), requiring two consecutive cleaving steps starting from preproadrenomedullin, an initial preprohormone. This preproadrenomedullin is converted into proadrenomedullin by cleavage of the signal peptide, so that three vasoactive peptides (ADM, the aminotemrinal peptide of proadrenomedullin (PAMP), and adrenotensin) as well as a region without known activity, entitled MR-proADM, remain [
95]. MR-proADM derives from the final proADM molecule in a ratio of 1:1 with ADM and therefore represents the levels and activity of ADM, which is almost inaccessible for biochemical analysis [
95]. Concerning its clinical usefulness, MR-proADM is known to be of diagnostic value for the discrimination of critically ill patients with all-cause sepsis (independent of the underlying microbial pathogen) from those without an infectious stimulus. Within this context, MR-proADM can either be used alone or in combination with other biomarkers (e.g., PCT) [
96]. Increased levels of MR-proADM (in parallel to increased ADM levels) were also shown to be associated with disease severity and the magnitude of organ failure, so that MR-proADM has proven to be of prognostic value in septic patients [
97]. This prognostic value of MR-proADM was further highlighted by a recently published prospective, observational study by Andaluz-Ojeda et al., in which MR-proADM was the only biomarker to predict sepsis mortality in all severity groups independent of the degree of organ failure (according to the Sequential Organ Failure Assessment (SOFA) score) [
98]. Moreover, ADM and PAMP appear to play a critical role in the host defense against systemic infections. Accordingly, expression of ADM is enhanced in connection with a polymicrobial insult, thus reaching adequate mucosal levels in order to serve as a local antimicrobial peptide [
99]. Both, ADM and PAMP have shown to be active against Gram-negative and Gram-positive bacteria as well as
Candida albicans [
100,
101,
102,
103,
104] and ADM was not only identified as the earliest target gene in response to epithelial infections with
Candida spp. within our recent experimental investigation but also in a recent study from Kuhbacher and colleagues after 24 h of infection [
105]. We thus conclude that (1) MR-proADM represents a key mediator of immunity in the context of candidiasis and (2) plasma level measurements of MR-proADM are therefore a suitable tool for the diagnosis of patients suffering from an invasive fungal infection. This is further supported by the investigation of Angeletti et al., in which MR-proADM levels were shown to be superior to PCT with regard to the identification of patients suffering from Gram-positive sepsis as well as yeast sepsis [
96]. Therefore, the routine use of MR-proADM measurements in patients at a high risk of an invasive fungal infection should therefore be taken into account.