Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay.

Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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.

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.

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata.

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

Cryptococcose disseminée: Manifestation inaugurale d'un SIDA [Disseminated cryptococcosis as inaugural manifestation of AIDS]

Disseminated cryptococcal disease is typically seen in patients with HIV infection. We report here the evolution of a patient with disseminated cryptococcosis whose treatment failed after ten weeks of induction therapy with amphotericin B. This case illustrates the importance of careful initial evaluation, and close clinical follow-up of these patients who are at risk of developing other opportunistic infections and drug-related complications.

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.

Safety concerns about CCR5 as an antiviral target.

PURPOSE OF REVIEW: Clinical trials of CCR5 antagonists attest to their efficacy and tolerance in HIV treatment. However, there has been debate on their long-term safety because of the role of CCR5 in innate immunity. This review highlights gaps in our understanding of epidemiology of infections that are modulated by CCR5, in particular, in HIV-infected individuals. RECENT FINDINGS: In the mouse model, CCR5 has a role in the response against pathogens as diverse as Toxoplama gondii, West Nile virus, Mycobacterium tuberculosis, herpes simplex virus, Trypanosoma cruzi, Cryptococcus neoformans, Chlamydia trachomatis, Listeria, and plasmodia. In human cohorts, individuals carrying the defective CCR5Delta32 allele present an increased susceptibility to flavivirus (West Nile virus and tickborne encephalitis virus). The selective pressures that led to the spread of loss-of-function CCR5 mutations in humans (CCR5Delta32), and in mangabeys (CCR5Delta24) are not understood. SUMMARY: The recent availability of CCR5 antagonists has raised concern that genetic, biological, or chemical CCR5 knockout, although beneficial against some pathogens (i.e. HIV), could be deleterious for other processes implicated in pathogen response. The consequences of long-term pharmaceutical intervention on CCR5 should be carefully assessed through rigorous postmarketing surveillance.

The Aspergillus fumigatus septins play pleiotropic roles in septation, conidiation, and cell wall stress, but are dispensable for virulence.

Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA-E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model.