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

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

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

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

Continuous Decline of Toxoplasma gondii Seroprevalence in Hospital: A 1997–2014 Longitudinal Study in Paris, France

Background: The protozoan Toxoplasma gondii presents a risk for reactivation of latent cysts in immunocompromised patients. Anti-T. gondii antibodies are therefore usually screened before chemotherapy or transplantation to propose prophylactic measures against this parasite. We analyzed the results obtained in our hospital to study the epidemiological trend of T. gondii infection.Methods: We collected all the anti-Toxoplasma antibody titers from January 1, 1997 to December 31, 2013 using the Platelia IgG ELISA assay (Bio-Rad). The results were classified as positive when titers reached a concentration of ≥10 UI/ml. Only the first result obtained at entry for each patient was considered. T. gondii seroprevalence was estimated using a multivariate logistic regression model accounting for age, sex, and year in which the sample was collected.Results: A total of 21,480 patient samples were analyzed. The seroprevalence continuously decreased over time, from 64.5% in 1997 to 54.7% in 2013 (i.e., an average of 1.3% per year, p < 0.001). The decrease was 5.0% per year for patients <20 years. After 2013, the model predicts that the seroprevalence would continuously decrease. We also observed a higher proportion of seropositive men than women in the 15- to 45-year-old group (58.5% versus 52.0%, p < 10-3).Conclusion: The overall seroprevalence of toxoplasmosis at our hospital showed an accelerating downward trend over 17 years. The reason for this continuous decline is likely associated with the lower parasite presence within meat. Thus, although young immunocompromised patients are increasingly less at risk of reactivation in the near future, older immunocompromised patients will remain at high risk of reactivation. The reasons of the higher prevalence in men remain to be explored

Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii's physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.This work was supported by research project PI14CIII/00045 from the Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III. CV is supported by research fellowships from the Fondo de Investigaciones Sanitarias of the Spanish Ministry of Economy and Competitiveness (FI12/00095).S

Molecular Demonstration of a Pneumocystis Outbreak in Stem Cell Transplant Patients: Evidence for Transmission in the Daycare Center

Pneumocystis jirovecii pneumonia (PCP) is a life-threatening infection in hematology. Although occasionally reported, the role of interhuman transmission of P. jirovecii in PCP, compared to that of reactivation, remains an unresolved question; the recommendation to isolate PCP patients in the hematology ward are not well evidence-based. Following an unexpected increase in the number of febrile pneumonia patients with P. jirovecii DNA detected in respiratory samples in our hematology ward, we explored 12 consecutive patients from November 2015 to May 2016. Genotyping of P jirovecii was performed using microsatellite markers. The frequency of simultaneous occupancy of these 12 patients in the same unit on the same day from 4 months prior to the first diagnosis was recorded. In three patients, the P. jirovecii genotype could not be determined because DNA was insufficient. One rare single genotype (Gt2) was found in four of the other nine, all allogeneic stem cell transplant recipients. The transmission map showed that these 4 patients had multiple opportunities to meet on the same day (median, 6.5; range, 4–10) at the daycare center. It was much less among the eight non-Gt2 patients (median, 1; range, 0–9; P = 0.048). This study, based on modern molecular technics, strongly suggests that interhuman transmission of P. jirovecii between allogeneic stem cell transplant recipients is possible. P. jirovecii DNA detected in respiratory specimens supports that isolation and respiratory precautions be recommended in such cases in the hematology ward

Molecular characterization and sterol profiles identify nonsynonymous mutations in ERG2 as a major mechanism conferring reduced susceptibility to amphotericin B in candida kefyr

The molecular basis of reduced susceptibility to amphotericin B (rs-AMB) among any yeasts is poorly defined. Genetic alterations in genes involved in ergosterol biosynthesis and total cell sterols were investigated among clinical Candida kefyr isolates. C. kefyr isolates (n = 81) obtained from 74 patients in Kuwait and identified by phenotypic and molecular methods were analyzed. An Etest was initially used to identify isolates with rs-AMB. Specific mutations in ERG2 and ERG6 involved in ergosterol biosynthesis were detected by PCR sequencing. Twelve selected isolates were also tested by the SensiTitre Yeast One (SYO), and total cell sterols were evaluated by gas chromatography-mass spectrometry and ERG3 and ERG11 sequencing. Eight isolates from 8 patients showed rs-AMB by Etest, including 2 isolates with additional resistance to fluconazole or to all three antifungals. SYO correctly identified 8 of 8 rs-AMB isolates. A nonsynonymous mutation in ERG2 was detected in 6 of 8 rs-AMB isolates but also in 3 of 73 isolates with a wild-type AMB pattern. One rs-AMB isolate contained a deletion (frameshift) mutation in ERG2. One or more nonsynonymous mutations was detected in ERG6 in 11 of 81 isolates with the rs-AMB or wild-type AMB pattern. Among 12 selected isolates, 2 and 2 isolates contained a nonsynonymous mutation(s) in ERG3 and ERG11, respectively. Ergosterol was undetectable in 7 of 8 rs-AMB isolates, and the total cell sterol profiles were consistent with loss of ERG2 function in 6 rs-AMB isolates and loss of ERG3 activity in another rs-AMB isolate. Our data showed that ERG2 is a major target conferring rs-AMB in clinical C. kefyr isolates