Recent advances in managing HIV-associated cryptococcal meningitis

The recent development of highly sensitive and specific point-of-care tests has made it possible to diagnose HIV-associated cryptococcal meningitis within minutes. However, diagnostic advances have not been matched by new antifungal drugs and treatment still relies on old off-patent drugs: amphotericin B, flucytosine and fluconazole. Cryptococcal meningitis treatment is divided in three phases: induction, consolidation and maintenance. The induction phase, aimed at drastically reducing cerebrospinal fluid fungal burden, is key for patient survival. The major challenge in cryptococcal meningitis management has been the optimisation of induction phase treatment using the limited number of available medications, and major progress has recently been made. In this review, we summarise data from key trials which form the basis of current treatment recommendations for HIV-associated cryptococcal meningitis

Deciphering the unusual HLA-A2/Melan-A/MART-1-specific TCR repertoire in humans.

The Melan-A/MART-1(26-35) antigenic peptide is one of the best studied human tumor-associated antigens. It is expressed in healthy melanocytes and malignant melanoma and is recognized by CD8(+) T cells in the context of the MHC class I molecule HLA-A*0201. While an unusually large repertoire of CD8(+) T cells specific for this antigen has been documented, the reasons for its generation have remained elusive. In this issue of the European Journal of Immunology, Pinto et al. [Eur. J. Immunol. 2014. 44: 2811-2821] uncover one important mechanism by comparing the thymic expression of the Melan-A gene to that in the melanocyte lineage. This study shows that medullary thymic epithelial cells (mTECs) dominantly express a truncated Melan-A transcript, the product of misinitiation of transcription. Consequently, the protein product in mTECs lacks the immunodominant epitope spanning residues 26-35, thus precluding central tolerance to this antigen. In contrast, melanocytes and melanoma tumor cells express almost exclusively the full-length Melan-A transcript, thus providing the target antigen for efficient recognition by HLA-A2-restricted CD8(+) T cells. The frequency of these alternative gene transcription modes may be more common than previously appreciated and may represent an important factor modulating the efficiency of central tolerance induction in the thymus

Dynamics of Cryptococcus neoformans-Macrophage Interactions Reveal that Fungal Background Influences Outcome during Cryptococcal Meningoencephalitis in Humans

Cryptococcosis is a multifaceted fungal infection with variable clinical presentation and outcome. As in many infectious diseases, this variability is commonly assigned to host factors. To investigate whether the diversity of Cryptococcus neoformans clinical (ClinCn) isolates influences the interaction with host cells and the clinical outcome, we developed and validated new quantitative assays using flow cytometry and J774 macrophages. The phenotype of ClinCn-macrophage interactions was determined for 54 ClinCn isolates recovered from cerebrospinal fluids (CSF) from 54 unrelated patients, based on phagocytic index (PI) and 2-h and 48-h intracellular proliferation indexes (IPH2 and IPH48, respectively). Their phenotypes were highly variable. Isolates harboring low PI/low IPH2 and high PI/high IPH2 values were associated with nonsterilization of CSF at week 2 and death at month 3, respectively. A subset of 9 ClinCn isolates with different phenotypes exhibited variable virulence in mice and displayed intramacrophagic expression levels of the LAC1, APP1, VAD1, IPC1, PLB1, and COX1 genes that were highly variable among the isolates and correlated with IPH48. Variation in the expression of virulence factors is thus shown here to depend on not only experimental conditions but also fungal background. These results suggest that, in addition to host factors, the patient’s outcome can be related to fungal determinants. Deciphering the molecular events involved in C. neoformans fate inside host cells is crucial for our understanding of cryptococcosis pathogenesis

Dynamique de l'adaptation de Cryptococcus neoformans à l'hôte

Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This pathogen is a facultative intracellular organism. Interaction with immune cells including monocytes/macrophages lineage and dendritic cells are of major importance in the natural history of the infection. In humans, the pathophysiology of the infection evolves in three steps (i) primoinfection in childhood, (ii) dormancy, demonstrated from epidemiological and genotypic data in the lab few years ago (Garcia-Hermoso et al. 1999), (iii) reactivation upon immunosuppression. In terms of disease, clinical presentation and outcome of cryptococcosis are known to be diverse among individuals even those sharing the same underlying diseases. To address the question of the impact of fungal diversity on the natural course of the infection at the macrophage-level (standardized model of yeasts/ j774 macrophages in vitro interaction), murine model-level (murine model of cryptococcosis in OF1 outbred mice) and human-level (CryptoA/D database), we studied (i) the diversity of C. neoformans/macrophages interactions using well characterized clinical isolates (ii) the correlation between in vitro phenotype of the isolate and clinical outcome in humans (iii) the diversity of adaptation to the host. We developed new assays and new tools using flow cytometry I (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), gene expression analysis (single-cell quantitative real time PCR) to overcome technical issues. We found high variation in phagocytic, 2 hours-, 48hours-intracellular proliferation indexes among the 54 ClinCn compared to H99. No correlation with the genotype was observed. The lack of sterilization at week 2 despite active antifungal therapy was significantly associated with a lower phagocytic index, whereas treatment failure at month 3 and death from cryptococcosis were significantly related to a higher 2 hours-intracellular proliferation. Among 9 selected clinical isolates compared to H99, (i) the virulence in mice was significantly different, intracellular expression of some virulence factors correlated with (ii) intracellular proliferation and (iii) phagocytic indexes. With a focus on multiplication and stress response and considering the H99 reference strain, we observed the appearance of various populations of yeasts during mice and macrophage infections. After sorting yeasts populations, we observed that a specific one was less prone and dependent of serum to grow compared to the other p'opulations. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. We found a high diversity of C. neoformans upon interaction with macrophages considering 54 clinical isolates in correlation with clinical outcome in humans, but also a considerable adaptation to host in our two models considering the reference strains H99. We observed also even more diversity of fungal adaptation to host when clinical isolates were considered. Ail together, these data suggest that cryptococcosis and fungal disease in general could be more complex diseases since diversity, plasticity and adaptation of the fungal organism to hosts is high and heterogeneous.La cryptococcose est une infection opportuniste causée par la levure ubiquitaire Cryptococcus neoformans. L'interaction avec les macrophages est importante pour le développement de la maladie chez l'homme. La physiopathologie évolue en trois étapes: (i) primo-infection, (ii) dormance, (iii) réactivation en cas d'immunodépression cellulaire. La présentation clinique et l'évolution de la cryptococcose sont variables en fonction des individus. L'objectif de mon travail était de comprendre l'impact de la diversité de la levure sur l'évolution de l'infection. Nous avons étudiés (i) les souches cliniques et leur diversité d'interaction avec les macrophages; (ii) la corrélation entre le phénotype in vitro et l'évolution clinique de l'infection chez l'homme; (iii) la diversité d'adaptation à l'hôte. Ainsi, nous avons développés de nouveaux outils (cytométrie de flux quantitative, microscopie en temps réel, single-cell quantitative PCR) pour permettre l'analyse de populations de levures rares. Nous avons trouvés une grande variabilité d'interaction de 54 isolats cliniques avec le macrophage. L'échec mycologique et la mortalité chez l'homme était associés à des phénotypes d'interaction particuliers. A partir de l'analyse de 9 isolats cliniques, nous avons pu mettre en évidence que la virulence des isolats et l'expression de certains gènes de virulence connus étaient variables également. En nous focalisant sur la réponse au stress et la prolifération, nous avons observé l'apparition de différentes populations au cours de l'infection. Après tri des différentes populations, nous avons identifié une population ayant une faible réponse au stress et montré qu'elle était potentiellement dormante. Ces données suggèrent que la cryptococcose et les infections fongiques en générale sont des infections complexes résultants d'une grande diversité, plasticité et adaptation des champignons à l'hôte

Effect of Virulence Factors on the Photodynamic Inactivation of Cryptococcus neoformans

Opportunistic fungal pathogens may cause an array of superficial infections or serious invasive infections, especially in immunocompromised patients. Cryptococcus neoformans is a pathogen causing cryptococcosis in HIV/AIDS patients, but treatment is limited due to the relative lack of potent antifungal agents. Photodynamic inactivation (PDI) uses the combination of non-toxic dyes called photosensitizers and harmless visible light, which produces singlet oxygen and other reactive oxygen species that produce cell inactivation and death. We report the use of five structurally unrelated photosensitizers (methylene blue, Rose Bengal, selenium derivative of a Nile blue dye, a cationic fullerene and a conjugate between poly-L-lysine and chlorin(e6)) combined with appropriate wavelengths of light to inactivate C. neoformans. Mutants lacking capsule and laccase, and culture conditions that favoured melanin production were used to probe the mechanisms of PDI and the effect of virulence factors. The presence of cell wall, laccase and melanin tended to protect against PDI, but the choice of the appropriate photosensitizers and dosimetry was able to overcome this resistance.Fundação de Amparo à Pesquisa do Estado de São Paulo (2010/13313–9

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

Background: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. Objectives: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. Methods: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. Results: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. Conclusion: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.This work was supported by research project PI21CIII/00007 from Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos IIIS

In vitro and in vivo antifungal profile of a novel and long acting inhaled azole, PC945, on Aspergillus fumigatus infection

The profile of PC945, a novel triazole antifungal, designed for administration via inhalation, hasbeen assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tight-binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B)activity.In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945, and thenwashed, PC945 was found to be quickly absorbed into both target and non-target cells and toproduce persistent antifungal effects. In temporarily neutropenic immunocompromised miceinfected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treatedintranasally with PC945 at 0.56 μg/mouse, while posaconazole showed similar effects (44%) at14 μg/mouse. This profile affirms that topical treatment with PC945 should provide potentantifungal activity in the lung

Recent advances in the understanding and management of mucormycosis [version 1; referees: 2 approved]

Mucormycoses were difficult-to-manage infections owing to limited diagnostic tools and therapeutic options. We review here advances in pathology understanding, diagnostic tools including computed tomography, and serum polymerase chain reaction and therapeutic options

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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