Defining responses to therapy and study outcomes in clinical trials of invasive fungal diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer consensus criteria.

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation.

Invasive opportunistic fungal diseases (IFDs) are important causes of morbidity and mortality in paediatric patients with cancer and those who have had an allogeneic haemopoietic stem-cell transplantation (HSCT). Apart from differences in underlying disorders and comorbidities relative to those of adults, IFDs in infants, children, and adolescents are unique with respect to their epidemiology, the usefulness of diagnostic methods, the pharmacology and dosing of antifungal agents, and the absence of interventional phase 3 clinical trials for guidance of evidence-based decisions. To better define the state of knowledge on IFDs in paediatric patients with cancer and allogeneic HSCT and to improve IFD diagnosis, prevention, and management, the Fourth European Conference on Infections in Leukaemia (ECIL-4) in 2011 convened a group that reviewed the scientific literature on IFDs and graded the available quality of evidence according to the Infectious Diseases Society of America grading system. The final considerations and recommendations of the group are summarised in this manuscript.

Application of the 2008 definitions for invasive fungal diseases to the trial comparing voriconazole versus amphotericin B for therapy of invasive aspergillosis: a collaborative study of the Mycoses Study Group (MSG 05) and the European Organization for Research and Treatment of Cancer Infectious Diseases Group.

BACKGROUND: Strict definition of invasive aspergillosis (IA) cases is required to allow precise conclusions about the efficacy of antifungal therapy. The Global Comparative Aspergillus Study (GCAS) compared voriconazole to amphotericin B (AmB) deoxycholate for the primary therapy of IA. Because predefined definitions used for this trial were substantially different from the consensus definitions proposed by the European Organization for Research and Treatment of Cancer/Mycoses Study Group in 2008, we recategorized the 379 episodes of the GCAS according to the later definitions. METHODS: The objectives were to assess the impact of the current definitions on the classification of the episodes and to provide comparative efficacy for probable/proven and possible IA in patients treated with either voriconazole or AmB. In addition to original data, we integrated the results of baseline galactomannan serum levels obtained from 249 (65.7%) frozen samples. The original response assessment was accepted unchanged. RESULTS: Recategorization allowed 59 proven, 178 probable, and 106 possible IA cases to be identified. A higher favorable 12-week response rate was obtained with voriconazole (54.7%) than with AmB (29.9%) (P < .0001). Survival was higher for voriconazole for mycologically documented (probable/proven) IA (70.2%) than with AmB (54.9%) (P = .010). Higher response rates were obtained in possible IA treated with voriconazole vs AmB with the same magnitude of difference (26.2%; 95% confidence interval [CI], 7.2%-45.3%) as in mycologically documented episodes (24.3%; 95% CI, 11.9%-36.7%), suggesting that possible cases are true IA. CONCLUSIONS: Recategorization resulted in a better identification of the episodes and confirmed the higher efficacy of voriconazole over AmB deoxycholate in mycologically documented IA.

ECIL-3 classical diagnostic procedures for the diagnosis of invasive fungal diseases in patients with leukaemia.

Invasive fungal diseases (IFDs) continue to cause considerable morbidity and mortality in patients with haematological malignancy. Diagnosis of IFD is difficult, with the sensitivity of the gold standard tests (culture and histopathology) often reported to be low, which may at least in part be due to sub-optimal sampling or subsequent handling in the routine microbiological laboratory. Therefore, a working group of the European Conference in Infections in Leukaemia was convened in 2009 with the task of reviewing the classical diagnostic procedures and providing recommendations for their optimal use. The recommendations were presented and approved at the ECIL-3 conference in September 2009. Although new serological and molecular tests are examined in separate papers, this review focuses on sample types, microscopy and culture procedures, antifungal susceptibility testing and imaging. The performance and limitations of these procedures are discussed and recommendations are provided on when and how to use them and how to interpret the results.

ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients.

As culture-based methods for the diagnosis of invasive fungal diseases (IFD) in leukemia and hematopoietic SCT patients have limited performance, non-culture methods are increasingly being used. The third European Conference on Infections in Leukemia (ECIL-3) meeting aimed at establishing evidence-based recommendations for the use of biological tests in adult patients, based on the grading system of the Infectious Diseases Society of America. The following biomarkers were investigated as screening tests: galactomannan (GM) for invasive aspergillosis (IA); β-glucan (BG) for invasive candidiasis (IC) and IA; Cryptococcus Ag for cryptococcosis; mannan (Mn) Ag/anti-mannan (A-Mn) Ab for IC, and PCR for IA. Testing for GM, Cryptococcus Ag and BG are included in the revised EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) consensus definitions for IFD. Strong evidence supports the use of GM in serum (A II), and Cryptococcus Ag in serum and cerebrospinal fluid (CSF) (A II). Evidence is moderate for BG detection in serum (B II), and the combined Mn/A-Mn testing in serum for hepatosplenic candidiasis (B III) and candidemia (C II). No recommendations were formulated for the use of PCR owing to a lack of standardization and clinical validation. Clinical utility of these markers for the early management of IFD should be further assessed in prospective randomized interventional studies.

Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.

BACKGROUND: Invasive fungal diseases are important causes of morbidity and mortality. Clarity and uniformity in defining these infections are important factors in improving the quality of clinical studies. A standard set of definitions strengthens the consistency and reproducibility of such studies. METHODS: After the introduction of the original European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group definitions, advances in diagnostic technology and the recognition of areas in need of improvement led to a revision of this document. The revision process started with a meeting of participants in 2003, to decide on the process and to draft the proposal. This was followed by several rounds of consultation until a final draft was approved in 2005. This was made available for 6 months to allow public comment, and then the manuscript was prepared and approved. RESULTS: The revised definitions retain the original classifications of "proven," "probable," and "possible" invasive fungal disease, but the definition of "probable" has been expanded, whereas the scope of the category "possible" has been diminished. The category of proven invasive fungal disease can apply to any patient, regardless of whether the patient is immunocompromised, whereas the probable and possible categories are proposed for immunocompromised patients only. CONCLUSIONS: These revised definitions of invasive fungal disease are intended to advance clinical and epidemiological research and may serve as a useful model for defining other infections in high-risk patients.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: adults with haematological malignancies and after haematopoietic stem cell transplantation (HCT).

Fungal diseases still play a major role in morbidity and mortality in patients with haematological malignancies, including those undergoing haematopoietic stem cell transplantation. Although Aspergillus and other filamentous fungal diseases remain a major concern, Candida infections are still a major cause of mortality. This part of the ESCMID guidelines focuses on this patient population and reviews pertaining to prophylaxis, empirical/pre-emptive and targeted therapy of Candida diseases. Anti-Candida prophylaxis is only recommended for patients receiving allogeneic stem cell transplantation. The authors recognize that the recommendations would have most likely been different if the purpose would have been prevention of all fungal infections (e.g. aspergillosis). In targeted treatment of candidaemia, recommendations for treatment are available for all echinocandins, that is anidulafungin (AI), caspofungin (AI) and micafungin (AI), although a warning for resistance is expressed. Liposomal amphotericin B received a BI recommendation due to higher number of reported adverse events in the trials. Amphotericin B deoxycholate should not be used (DII); and fluconazole was rated CI because of a change in epidemiology in some areas in Europe. Removal of central venous catheters is recommended during candidaemia but if catheter retention is a clinical necessity, treatment with an echinocandin is an option (CII(t) ). In chronic disseminated candidiasis therapy, recommendations are liposomal amphotericin B for 8 weeks (AIII), fluconazole for >3 months or other azoles (BIII). Granulocyte transfusions are only an option in desperate cases of patients with Candida disease and neutropenia (CIII).

Les maladies infectieuses ont encore un bel avenir [Infectious diseases have again a good future!].

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

Nonmolecular methods for the diagnosis of respiratory fungal infections.

Diagnosis of invasive fungal pneumonias by conventional culture methods is difficult to assess and often delayed. Nonmolecular fungal markers have emerged as an important adjunctive tool to support their diagnosis in combination with other clinical, radiologic, and microbiological criteria of invasive fungal diseases. Concerns about the sensitivity and specificity of some tests in different patient populations should lead to warnings about their widespread use. None can identify the emerging and particularly deadly fungal pathogens responsible for mucormycosis. The role of nonmolecular fungal markers should be better defined in combination with other microbiological and radiologic tools in preemptive antifungal strategies.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

The expression and impact of antifungal grooming in ants.

Parasites can cause extensive damage to animal societies in which many related individuals frequently interact. In response, social animals have evolved diverse individual and collective defences. Here, we measured the expression and efficiency of self-grooming and allo-grooming when workers of the ant Formica selysi were contaminated with spores of the fungal entomopathogen Metarhizium anisopliae. The amount of self-grooming increased in the presence of fungal spores, which shows that the ants are able to detect the risk of infection. In contrast, the amount of allo-grooming did not depend on fungal contamination. Workers groomed all nestmate workers that were re-introduced into their groups. The amount of allo-grooming towards noncontaminated individuals was higher when the group had been previously exposed to the pathogen. Allo-grooming decreased the number of fungal spores on the surface of contaminated workers, but did not prevent infection in the conditions tested (high dose of spores and late allo-grooming). The rate of disease transmission to groomers and other nestmates was extremely low. The systematic allo-grooming of all individuals returning to the colony, be they contaminated or not, is probably a simple but robust prophylactic defence preventing the spread of fungal diseases in insect societies.

Monitoring procalcitonin in febrile neutropenia: what is its utility for initial diagnosis of infection and reassessment in persistent Fever?

Background: Management of febrile neutropenic episodes (FE) is challenged by lacking microbiological and clinical documentation of infection. We aimed at evaluating the utility of monitoring blood procalcitonin (PCT) in FE for initial diagnosis of infection and reassessment in persistent fever.Methods: PCT kinetics was prospectively monitored in 194 consecutive FE (1771 blood samples): 65 microbiologically documented infections (MDI, 33.5%; 49 due to non-coagulase-negative staphylococci, non-CNS), 68 clinically documented infections (CDI, 35%; 39 deep-seated), and 61 fever of unexplained origin (FUO, 31.5%).Results: At fever onset median PCT was 190 pg/mL (range 30-26'800), without significant difference among MDI, CDI and FUO. PCT peak occurred on day 2 after onset of fever: non-CNS-MDI/deep-seated-CDI (656, 80-86350) vs. FUO (205, 33-771; p<0.001). PCT >500 pg/mL distinguished non-CNS-MDI/deep-seated-CDI from FUO with 56% sensitivity and 90% specificity. PCT was >500 pg/ml in only 10% of FUO (688, 570-771). A PCT peak >500 pg/mL (1196, 524-11950) occurred beyond 3 days of persistent fever in 17/21 (81%) invasive fungal diseases (IFD). This late PCT peak identified IFD with 81% sensitivity and 57% specificity and preceded diagnosis according to EORTC-MSG criteria in 41% of cases. In IFD responding to therapy, median days to PCT <500 pg/mL and defervescence were 5 (1-23) vs. 10 (3-22; p = 0.026), respectively.Conclusion: While procalcitonin is not useful for diagnosis of infection at onset of neutropenic fever, it may help to distinguish a minority of potentially severe infections among FUOs on day 2 after onset of fever. In persistent fever monitoring procalcitonin contributes to early diagnosis and follow-up of invasive mycoses

Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis.

A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms.

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.

Oral insecticidal activity of plant-associated pseudomonads.

Biocontrol pseudomonads are most known to protect plants from fungal diseases and to increase plant yield, while intriguing aspects on insecticidal activity have been discovered only recently. Here, we demonstrate that Fit toxin producing pseudomonads, in contrast to a naturally Fit-deficient strain, exhibit potent oral activity against larvae of Spodoptera littoralis, Heliothis virescens and Plutella xylostella, all major insect pests of agricultural crops. Spraying plant leaves with suspensions containing only 1000 Pseudomonas cells per ml was sufficient to kill 70-80% of Spodoptera and Heliothis larvae. Monitoring survival kinetics and bacterial titres in parallel, we demonstrate that Pseudomonas fluorescens CHA0 and Pseudomonas chlororaphis PCL1391, two bacteria harbouring the Fit gene cluster colonize and kill insects via oral infection. Using Fit mutants of CHA0 and PCL1391, we show that production of the Fit toxin contributes substantially to oral insecticidal activity. Furthermore, the global regulator GacA is required for full insecticidal activity. Our findings demonstrate the lethal oral activity of two root-colonizing pseudomonads so far known as potent antagonists of fungal plant pathogens. This adds insecticidal activity to the existing biocontrol repertoire of these bacteria and opens new perspectives for applications in crop pest control and in research on their ecological behaviour.