Epidemiology and outcome of fungemia in a cancer Cohort of the Infectious Diseases Group (IDG) of the European Organization for Research and Treatment of Cancer (EORTC 65031).

BACKGROUND: Anti-cancer treatment and the cancer population have evolved since the last European Organisation for Research and Treatment of Cancer (EORTC) fungemia survey, and there are few recent large epidemiological studies. METHODS: This was a prospective cohort study including 145 030 admissions of patients with cancer from 13 EORTC centers. Incidence, clinical characteristics, and outcome of fungemia were analyzed. RESULTS: Fungemia occurred in 333 (0.23%; 95% confidence interval [CI], .21-.26) patients, ranging from 0.15% in patients with solid tumors to 1.55% in hematopoietic stem cell transplantation recipients. In 297 evaluable patients age ranged from 17 to 88 years (median 56 years), 144 (48%) patients were female, 165 (56%) had solid tumors, and 140 (47%) had hematological malignancies. Fungemia including polymicrobial infection was due to: Candida spp. in 267 (90%), C. albicans in 128 (48%), and other Candida spp. in 145 (54%) patients. Favorable overall response was achieved in 113 (46.5%) patients by week 2. After 4 weeks, the survival rate was 64% (95% CI, 59%-70%) and was not significantly different between Candida spp. Multivariable logistic regression identified baseline septic shock (odds ratio [OR] 3.04, 95% CI, 1.22-7.58) and tachypnoea as poor prognostic factors (OR 2.95, 95% CI, 1.66-5.24), while antifungal prophylaxis prior to fungemia (OR 0.20, 95% CI, .06-.62) and remission of underlying cancer (OR, 0.18; 95% CI, .06-.50) were protective. CONCLUSIONS: Fungemia, mostly due to Candida spp., was rare in cancer patients from EORTC centers but was associated with substantial mortality. Antifungal prophylaxis and remission of cancer predicted better survival.

Tipping the balance both ways: drug resistance and virulence in Candida glabrata.

Among existing fungal pathogens, Candida glabrata is outstanding in its capacity to rapidly develop resistance to currently used antifungal agents. Resistance to the class of azoles, which are still widely used agents, varies in proportion (from 5 to 20%) depending on geographical area. Moreover, resistance to the class of echinocandins, which was introduced in the late 1990s, is rising in several institutions. The recent emergence of isolates with acquired resistance to both classes of agents is a major concern since alternative therapeutic options are scarce. Although considered less pathogenic than C. albicans, C. glabrata has still evolved specific virulence traits enabling its survival and propagation in colonized and infected hosts. Development of drug resistance is usually associated with fitness costs, and this notion is documented across several microbial species. Interestingly, azole resistance in C. glabrata has revealed the opposite. Experimental models of infection showed enhanced virulence of azole-resistant isolates. Moreover, azole resistance could be associated with specific changes in adherence properties to epithelial cells or innate immunity cells (macrophages), both of which contribute to virulence changes. Here we will summarize the current knowledge on C. glabrata drug resistance and also discuss the consequences of drug resistance acquisition on the balance between C. glabrata and its hosts.

Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus.

Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.

Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations.

OBJECTIVES: The objective of this study was to characterize the underlying molecular mechanisms in consecutive clinical Candida albicans isolates from a single patient displaying stepwise-acquired multidrug resistance. METHODS: Nine clinical isolates (P-1 to P-9) were susceptibility tested by EUCAST EDef 7.2 and Etest. P-4, P-5, P-7, P-8 and P-9 were available for further studies. Relatedness was evaluated by MLST. Additional genes were analysed by sequencing (including FKS1, ERG11, ERG2 and TAC1) and gene expression by quantitative PCR (CDR1, CDR2 and ERG11). UV-spectrophotometry and GC-MS were used for sterol analyses. In vivo virulence was determined in the insect model Galleria mellonella and evaluated by log-rank Mantel-Cox tests. RESULTS: P-1 + P-2 were susceptible, P-3 + P-4 fluconazole resistant, P-5 pan-azole resistant, P-6 + P-7 pan-azole and echinocandin resistant and P-8 + P-9 MDR. MLST supported genetic relatedness among clinical isolates. P-4 harboured four changes in Erg11 (E266D, G307S, G450E and V488I), increased expression of ERG11 and CDR2 and a change in Tac1 (R688Q). P-5, P-7, P-8 and P-9 had an additional change in Erg11 (A61E), increased expression of CDR1, CDR2 and ERG11 (except for P-7) and a different amino acid change in Tac1 (R673L). Echinocandin-resistant isolates harboured the Fks1 S645P alteration. Polyene-resistant P-8 + P-9 lacked ergosterol and harboured a frameshift mutation in ERG2 (F105SfsX23). Virulence was attenuated (but equivalent) in the clinical isolates, but higher than in the azole- and echinocandin-resistant unrelated control strain. CONCLUSIONS: C. albicans demonstrates a diverse capacity to adapt to antifungal exposure. Potentially novel resistance-inducing mutations in TAC1, ERG11 and ERG2 require independent validation.

Potential Microbiological Effects of Higher Dosing of Echinocandins.

The antifungal "paradoxical effect" has been described as the reversal of growth inhibition at high doses of echinocandins, most usually caspofungin. This microbiological effect appears to be a cellular compensatory response to cell wall damage, resulting in alteration of cell wall content and structure as well as fungal morphology and growth. In vitro studies demonstrate this reproducible effect in a certain percentage of fungal isolates, but animal model and clinical studies are less consistent. The calcineurin and Hsp90 cell signaling pathways appear to play a major role in regulating these cellular and structural changes. Regardless of the clinical relevance of this paradoxical growth effect, understanding the specific actions of echinocandins is paramount to optimizing their use at either standard or higher dosing schemes, as well as developing future improvements in our antifungal arsenal.

The Role of Biomarkers for Starting Antifungals in the Intensive Care Unit

Invasive candidiasis is associated with high mortality rates (35% to 60%), similar to the range reported for septic shock. The most common types include candidemia, frequently observed in immunocompromised patients, and noncandidemic systemic candidiasis, which constitutes the majority of cases in critically ill patients. However, they are difficult to prove and a definite diagnosis usually occurs late in the course of the disease, thus contributing to their bad prognosis. Early empirical treatment improves the prognosis and currently relies on the positive predictive value (PPV) of risk-assessment strategies (colonization index, Candida score, predictive rules) based on combinations of risk factors, but it may have also largely contributed to the overuse of antifungal agents in critically ill patients. In this context, non- culture-based diagnostic methods, including specific and nonspecific biomarkers, may significantly improve the diagnosis of invasive candidiasis. Candida DNA and mannan antigen/antimannan antibodies are of limited interest for the diagnosis of invasive candidiasis as they fail to identify noncandidemic systemic candidiasis, despite early positivity in candidemic patients. The utility of 1,3-beta-D-glucan (b-D-glucan), a panfungal cell wall antigen, has been demonstrated for the diagnosis of fungal infections in immunocompromised patients. Preliminary data suggest that it is also detectable early in critically ill patients developing noncandidemic systemic candidiasis. To take advantage of the high negative predictive value of risk-assessment strategies and the early increase in specific fungal biomarkers in high-risk patients, we propose a practical 2-step approach to improve the selection of patients susceptible to benefit from empirical antifungal treatment.

Novel Approaches for Fungal Transcriptomics from Host Samples.

Candida albicans adaptation to the host requires a profound reprogramming of the fungal transcriptome as compared to in vitro laboratory conditions. A detailed knowledge of the C. albicans transcriptome during the infection process is necessary in order to understand which of the fungal genes are important for host adaptation. Such genes could be thought of as potential targets for antifungal therapy. The acquisition of the C. albicans transcriptome is, however, technically challenging due to the low proportion of fungal RNA in host tissues. Two emerging technologies were used recently to circumvent this problem. One consists of the detection of low abundance fungal RNA using capture and reporter gene probes which is followed by emission and quantification of resulting fluorescent signals (nanoString). The other is based first on the capture of fungal RNA by short biotinylated oligonucleotide baits covering the C. albicans ORFome permitting fungal RNA purification. Next, the enriched fungal RNA is amplified and subjected to RNA sequencing (RNA-seq). Here we detail these two transcriptome approaches and discuss their advantages and limitations and future perspectives in microbial transcriptomics from host material.