Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients.

Objectives: Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) suffer from chronic candidosis caused mainly by Candida albicans, and repeated courses of azole antifungals have led to the development of resistance in the APECED patient population in Finland. The aim of our study was to address whether the patients are persistently colonized with the same or genetically closely related strains, whether epidemic strains are present and which molecular mechanisms account for azole resistance. Methods: Sets of C. albicans (n?=?19) isolates from nine APECED patients reported with decreased susceptibility to fluconazole isolated up to 9 years apart were included. The strains were typed by multilocus sequence typing. CDR1/2, MDR1 and ERG11 mRNA expression was analysed by northern blotting and Cdr1, Cdr2 and Mdr1 protein expression by western blotting, and TAC1 and ERG11 genes were sequenced. Results: All seven patients with multiple C. albicans isolates analysed were persistently colonized with the same or a genetically closely related strain for a mean of 5 years. All patients were colonized with different strains and no epidemic strains were found. The major molecular mechanisms behind the azole resistance were mutations in TAC1 contributing to overexpression of CDR1 and CDR2. Six new TAC1 mutations were found, one of which (N740S) is likely to be a gain-of-function mutation. Most isolates were found to have gained multiple TAC1 and ERG11 point mutations. Conclusions: Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations occur within strains, leading to the development of azole-resistant isolates.

IL1B and DEFB1 Polymorphisms Increase Susceptibility to Invasive Mold Infection After Solid-Organ Transplantation.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in immune genes have been associated with susceptibility to invasive mold infection (IMI) among hematopoietic stem cell but not solid-organ transplant (SOT) recipients. METHODS: Twenty-four SNPs from systematically selected genes were genotyped among 1101 SOT recipients (715 kidney transplant recipients, 190 liver transplant recipients, 102 lung transplant recipients, 79 heart transplant recipients, and 15 recipients of other transplants) from the Swiss Transplant Cohort Study. Association between SNPs and the end point were assessed by log-rank test and Cox regression models. Cytokine production upon Aspergillus stimulation was measured by enzyme-linked immunosorbent assay in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and correlated with relevant genotypes. RESULTS: Mold colonization (n = 45) and proven/probable IMI (n = 26) were associated with polymorphisms in the genes encoding interleukin 1β (IL1B; rs16944; recessive mode, P = .001 for colonization and P = .00005 for IMI, by the log-rank test), interleukin 1 receptor antagonist (IL1RN; rs419598; P = .01 and P = .02, respectively), and β-defensin 1 (DEFB1; rs1800972; P = .001 and P = .0002, respectively). The associations with IL1B and DEFB1 remained significant in a multivariate regression model (P = .002 for IL1B rs16944; P = .01 for DEFB1 rs1800972). The presence of 2 copies of the rare allele of rs16944 or rs419598 was associated with reduced Aspergillus-induced interleukin 1β and tumor necrosis factor α secretion by PBMCs. CONCLUSIONS: Functional polymorphisms in IL1B and DEFB1 influence susceptibility to mold infection in SOT recipients. This observation may contribute to individual risk stratification.

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

Antifungal activity against planktonic and biofilm Candida albicans in an experimental model of foreign-body infection.

OBJECTIVES: The treatment of Candida implant-associated infections remains challenging. We investigated the antifungal activity against planktonic and biofilm Candida albicans in a foreign-body infection model. METHODS: Teflon cages were subcutaneously implanted in guinea pigs, infected with C. albicans (ATCC 90028). Animals were treated intraperitoneally 12 h after infection for 4 days once daily with saline, fluconazole (16 mg/kg), amphotericin B (2.5 mg/kg), caspofungin (2.5 mg/kg) or anidulafungin (20 mg/kg). Planktonic Candida was quantified, the clearance rate and cure rate determined. RESULTS: In untreated animals, planktonic Candida was cleared from cage fluid in 25% (infected with 4.5 × 10(3) CFU/cage), 8% (infected with 4.8 × 10(4) CFU/cage) and 0% (infected with 6.2 × 10(5) CFU/cage). Candida biofilm persisted on all explanted cages. Compared to untreated controls, caspofungin reduced the number of planktonic C. albicans to 0.22 and 0.0 CFU/ml, respectively, and anidulafungin to 0.11 and 0.13 CFU/ml, respectively. Fluconazole cured 2/12 cages (17%), amphotericin B and anidulafungin 1/12 cages (8%) and caspofungin 3/12 cages (25%). CONCLUSION: Echinocandins showed superior activity against planktonic C. albicans. Caspofungin showed the highest cure rate of C. albicans biofilm. However, no antifungal exceeded 25% cure rate, demonstrating the difficulty of eradicating Candida biofilms from implants.

Interleukin-33 safeguards neutrophils in sepsis.

Cytokines have a fundamental role in orchestrating innate immune responses to bacterial infections. Interleukin-33 (IL-33) is now shown to protect from sepsis by promoting neutrophil influx into the focus of infection

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.

OBJECTIVE: To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," published in 2004. DESIGN: Modified Delphi method with a consensus conference of 55 international experts, several subsequent meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. This process was conducted independently of any industry funding. METHODS: We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations. A strong recommendation (1) indicates that an intervention's desirable effects clearly outweigh its undesirable effects (risk, burden, cost) or clearly do not. Weak recommendations (2) indicate that the tradeoff between desirable and undesirable effects is less clear. The grade of strong or weak is considered of greater clinical importance than a difference in letter level of quality of evidence. In areas without complete agreement, a formal process of resolution was developed and applied. Recommendations are grouped into those directly targeting severe sepsis, recommendations targeting general care of the critically ill patient that are considered high priority in severe sepsis, and pediatric considerations. RESULTS: Key recommendations, listed by category, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm potential source of infection (1C); administration of broad-spectrum antibiotic therapy within 1 hr of diagnosis of septic shock (1B) and severe sepsis without septic shock (1D); reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate (1C); a usual 7-10 days of antibiotic therapy guided by clinical response (1D); source control with attention to the balance of risks and benefits of the chosen method (1C); administration of either crystalloid or colloid fluid resuscitation (1B); fluid challenge to restore mean circulating filling pressure (1C); reduction in rate of fluid administration with rising filing pressures and no improvement in tissue perfusion (1D); vasopressor preference for norepinephrine or dopamine to maintain an initial target of mean arterial pressure > or = 65 mm Hg (1C); dobutamine inotropic therapy when cardiac output remains low despite fluid resuscitation and combined inotropic/vasopressor therapy (1C); stress-dose steroid therapy given only in septic shock after blood pressure is identified to be poorly responsive to fluid and vasopressor therapy (2C); recombinant activated protein C in patients with severe sepsis and clinical assessment of high risk for death (2B except 2C for postoperative patients). In the absence of tissue hypoperfusion, coronary artery disease, or acute hemorrhage, target a hemoglobin of 7-9 g/dL (1B); a low tidal volume (1B) and limitation of inspiratory plateau pressure strategy (1C) for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure in acute lung injury (1C); head of bed elevation in mechanically ventilated patients unless contraindicated (1B); avoiding routine use of pulmonary artery catheters in ALI/ARDS (1A); to decrease days of mechanical ventilation and ICU length of stay, a conservative fluid strategy for patients with established ALI/ARDS who are not in shock (1C); protocols for weaning and sedation/analgesia (1B); using either intermittent bolus sedation or continuous infusion sedation with daily interruptions or lightening (1B); avoidance of neuromuscular blockers, if at all possible (1B); institution of glycemic control (1B), targeting a blood glucose < 150 mg/dL after initial stabilization (2C); equivalency of continuous veno-veno hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1A); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding using H2 blockers (1A) or proton pump inhibitors (1B); and consideration of limitation of support where appropriate (1D). Recommendations specific to pediatric severe sepsis include greater use of physical examination therapeutic end points (2C); dopamine as the first drug of choice for hypotension (2C); steroids only in children with suspected or proven adrenal insufficiency (2C); and a recommendation against the use of recombinant activated protein C in children (1B). CONCLUSIONS: There was strong agreement among a large cohort of international experts regarding many level 1 recommendations for the best current care of patients with severe sepsis. Evidenced-based recommendations regarding the acute management of sepsis and septic shock are the first step toward improved outcomes for this important group of critically ill patients.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: developing European guidelines in clinical microbiology and infectious diseases.

The process to develop a guideline in a European setting remains a challenge. The ESCMID Fungal Infection Study Group (EFISG) successfully achieved this endeavour. After two face-to-face meetings, numerous telephone conferences, and email correspondence, an ESCMID task force (basically composed of members of the Society's Fungal Infection Study Group, EFISG) finalized the ESCMID diagnostic and management/therapeutic guideline for Candida diseases. By appreciating various patient populations at risk for Candida diseases, four subgroups were predefined, mainly ICU patients, paediatric, HIV/AIDS and patients with malignancies including haematopoietic stem cell transplantation. Besides treatment recommendations, the ESCMID guidelines provide guidance for diagnostic procedures. For the guidelines, questions were formulated to phrase the intention of a given recommendation, for example, outcome. The recommendation was the clinical intervention, which was graded by a score of A-D for the 'Strength of a recommendation'. The 'level of evidence' received a score of I-III. The author panel was approved by ESCMID, European Organisation for Research and Treatment of Cancer, European Group for Blood and Marrow Transplantation, European Society of Intensive Care Medicine and the European Confederation of Medical Mycology. The guidelines followed the framework of GRADE and Appraisal of Guidelines, Research, and Evaluation. The drafted guideline was presented at ECCMID 2011 and points of discussion occurring during that meeting were incorporated into the manuscripts. These ESCMID guidelines for the diagnosis and management of Candida diseases provide guidance for clinicians in their daily decision-making process.

Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans.

Several types of drugs currently used in clinical practice were screened in vitro for their potentiation of the antifungal effect of the fungistatic agent fluconazole (FLC) on Candida albicans. These drugs included inhibitors of multidrug efflux transporters, antimicrobial agents, antifungal agents, and membrane-active compounds with no antimicrobial activity, such as antiarrhythmic agents, proton pump inhibitors, and platelet aggregation inhibitors. Among the drugs tested in an agar disk diffusion assay, cyclosporine (Cy), which had no intrinsic antifungal activity, showed a potent antifungal effect in combination with FLC. In a checkerboard microtiter plate format, however, it was observed that the MIC of FLC, as classically defined by the NCCLS recommendations, was unchanged when FLC and Cy were combined. Nevertheless, if a different reading endpoint corresponding to the minimal fungicidal concentration needed to decrease viable counts by at least 3 logs in comparison to the growth control was chosen, the combination was synergistic (fractional inhibitory concentration index of &lt;1). This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of the trailing effect, which is the result of a residual growth at FLC concentrations greater than the MIC. The MIC-0 values of FLC and Cy tested alone in C. albicans were &gt;32 and &gt;10 microg/ml, respectively, and decreased to 0.5 and 0.625 microg/ml when the two drugs were combined. The combination of 0.625 microg of Cy per ml with supra-MICs of FLC resulted in a potent antifungal effect in time-kill curve experiments. This effect was fungicidal or fungistatic, depending on the C. albicans strain used. Since the Cy concentration effective in vitro is achievable in vivo, the combination of this agent with FLC represents an attractive perspective for the development of new management strategies for candidiasis.

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis.

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Quantifying uncertainty: physicians' estimates of infection in critically ill neonates and children.

To determine the diagnostic accuracy of physicians' prior probability estimates of serious infection in critically ill neonates and children, we conducted a prospective cohort study in 2 intensive care units. Using available clinical, laboratory, and radiographic information, 27 physicians provided 2567 probability estimates for 347 patients (follow-up rate, 92%). The median probability estimate of infection increased from 0% (i.e., no antibiotic treatment or diagnostic work-up for sepsis), to 2% on the day preceding initiation of antibiotic therapy, to 20% at initiation of antibiotic treatment (P&lt;.001). At initiation of treatment, predictions discriminated well between episodes subsequently classified as proven infection and episodes ultimately judged unlikely to be infection (area under the curve, 0.88). Physicians also showed a good ability to predict blood culture-positive sepsis (area under the curve, 0.77). Treatment and testing thresholds were derived from the provided predictions and treatment rates. Physicians' prognoses regarding the presence of serious infection were remarkably precise. Studies investigating the value of new tests for diagnosis of sepsis should establish that they add incremental value to physicians' judgment.

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.

Mécanismes physiopathologiques impliqués dans la dysfonction d'organes au cours du sepsis [Pathophysiological mechanisms of organ dysfunction in sepsis].

Sepsis is defined as the systemic inflammatory response to an infection. The occurrence of organ dysfunction increases the severity of sepsis. Complex interactions between multiple immunomodulating mediators and various cell populations, activated secondarily to the initial infectious insult, promote the development of organ dysfunction in sepsis. Although septic organ dysfunction has long been considered as the end result of chaotic, uncontrolled and deregulated inflammatory cascades, it might instead represent an adaptive response to avoid the occurrence of irreversible tissue damage and end-organ injury. In this article, we review the major mechanisms involved in organ dysfunction during sepsis, and also present the concept of organ dysfunction as an adaptive response to the septic process.

Systemic antibody responses to gut commensal bacteria during chronic HIV-1 infection.

BACKGROUND: Human systemic antibody responses to commensal microbiota are not well characterised during health and disease. Of particular interest is the analysis of their potential modulation caused by chronic HIV-1 infection which is associated with sustained enteropathy and systemic B cell disturbances reflected by impaired B cell responses and chronic B cell hyperactivity. The mechanisms underlying B cell hyperactivation and the specificities of the resulting hypergammaglobulinaemia are only poorly understood. METHODS: By a technique referred to as live bacterial FACS (fluorescence-activated cell sorting), the present study investigated systemic antibody responses to several gut and skin commensal bacteria as well as Candida albicans in longitudinal plasma and serum samples from healthy donors, chronic HIV-1-infected individuals with or without diarrhoea and patients with inflammatory bowel disease (IBD). RESULTS: The data show that systemic antibody responses to the commensal microbiota were abundantly present in humans and remained remarkably stable over years. Overall systemic antibody responses to gut commensal bacteria were not affected during chronic HIV-1 infection, with titres decreasing when normalised to elevated plasma immunoglobulin G (IgG) levels found in patients with HIV. In contrast, increases in the titres of high affinity antimicrobiota antibodies were detected in patients with IBD, demonstrating that conditions with known increased intestinal permeability and aberrant mutualism can induce changes in antibody titres observed in these assays. CONCLUSION: Neither HIV-associated enteropathy nor B cell dysfunction impact on the high-affinity systemic antibody responses to gut commensal bacteria. HIV-associated hypergammaglobulinaemia is therefore unlikely to be driven by induction of antimicrobiota antibodies.

Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans.

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.