The pathogenesis of cryptococcosis, including the events leading to the production of meningoencephalitis, is still largely unknown. Evidence of a transcellular passage of Cryptococcus neoformans ...across the blood-brain barrier (BBB) and subsequent BBB disruption exists, but the paracellular passage of free yeasts and the role of monocytes in yeast dissemination and brain invasion (Trojan horse method) remain uncertain. We used our model of disseminated cryptococcosis, in which crossing of the BBB starts 6 h after intravenous inoculation, to study paracellular passage of the BBB. We prepared bone marrow-derived monocytes (BMDM) infected in vitro with C. neoformans (BMDM yeasts) and free yeasts and measured fungal loads in tissues. (i) Spleen and lung CFU were >2-fold higher in mice treated with BMDM yeasts than in those treated with free yeasts for 1 and 24 h (P < 0.05), while brain CFU were increased (3.9 times) only at 24 h (P < 0.05). (ii) By comparing the kinetics of brain invasion in naïve mice and in mice with preestablished cryptococcosis, we found that CFU were lower in the latter case, except at 6 h, when CFU from mice inoculated with BMDM yeasts were comparable to those measured in naïve mice and 2.5-fold higher than those in mice with preestablished cryptococcosis who were inoculated with free yeasts. (iii) Late phagocyte depletion obtained by clodronate injection reduced disease severity and lowered the fungal burden by 40% in all organs studied. These results provide evidence for Trojan horse crossing of the BBB by C. neoformans, together with mechanisms involving free yeasts, and overall for a role of phagocytes in fungal dissemination.
To determine the epidemiology and trends of invasive fungal infections (IFIs) in France, we analyzed incidence, risk factors, and in-hospital death rates related to the most frequent IFIs registered ...in the national hospital discharge database during 2001-2010. The identified 35,876 IFI cases included candidemia (43.4%), Pneumocystis jirovecii pneumonia (26.1%), invasive aspergillosis (IA, 23.9%), cryptococcosis (5.2%), and mucormycosis (1.5%). The overall incidence was 5.9/100,000 cases/year and the mortality rate was 27.6%; both increased over the period (+1.5%, +2.9%/year, respectively). Incidences substantially increased for candidemia, IA, and mucormycosis. Pneumocystis jirovecii pneumonia incidence decreased among AIDS patients (-14.3%/year) but increased in non-HIV-infected patients (+13.3%/year). Candidemia and IA incidence was increased among patients with hematologic malignancies (>+4%/year) and those with chronic renal failure (>+10%/year). In-hospital deaths substantially increased in some groups, e.g., in those with hematologic malignancies. IFIs occur among a broad spectrum of non-HIV-infected patients and should be a major public health priority.
The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to ...100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.
Invasive yeast infections represent a major global public health issue, and only few antifungal agents are available. Azoles are one of the classes of antifungals used for treatment of invasive ...candidiasis. The determination of antifungal susceptibility profiles using standardized methods is important to identify resistant isolates and to uncover the potential emergence of intrinsically resistant species. Here, we report data on 9,319 clinical isolates belonging to 40 pathogenic yeast species recovered in France over 17 years. The antifungal susceptibility profiles were all determined at the National Reference Center for Invasive Mycoses and Antifungals based on the EUCAST broth microdilution method. The centralized collection and analysis allowed us to describe the trends of azole susceptibility of isolates belonging to common species, confirming the high susceptibility for
(
= 3,295),
(
= 641), and
(
= 820) and decreased susceptibility for
(
= 1,274) and
(
= 343). These profiles also provide interesting data concerning azole susceptibility of
species complex, showing comparable MIC distributions for the three species but lower MIC
s and MIC
s for serotype D (
= 208) compared to serotype A (
= 949) and AD hybrids (
= 177). Finally, these data provide useful information for rare and/or emerging species, such as
(
= 221),
(
= 184),
complex (
= 150),
complex (
= 87),
(
= 55), and
(
= 36).
Purpose
To analyze trends in incidence and mortality of candidemia in intensive care units (ICUs) vs. non-ICU hospitalized patients and to determine risk factors for infection by specific species and ...for death.
Methods
Active hospital-based surveillance program of incident episodes of candidemia due to common species in 24 tertiary care hospitals in the Paris area, France between October 2002 and September 2010.
Results
Among 2,507 adult cases included, 2,571
Candida
isolates were collected and species were
C. albicans
(56 %),
C. glabrata
(18.6 %),
C. parapsilosis
(11.5 %),
C. tropicalis
(9.3 %),
C. krusei
(2.9 %), and
C. kefyr
(1.8 %). Candidemia occurred in ICU in 1,206 patients (48.1 %). When comparing ICU vs. non-ICU patients, the former had significantly more frequent surgery during the past 30 days, were more often preexposed to fluconazole and treated with echinocandin, and were less frequently infected with
C. parapsilosis
. Risk factors and age remained unchanged during the study period. A significant increased incidence in the overall population and ICU was found. The odds of being infected with a given species in ICU was influenced by risk factors and preexposure to fluconazole and caspofungin. Echinocandins initial therapy increased over time in ICU (4.6 % first year of study, to 48.5 % last year of study,
p
< 0.0001). ICU patients had a higher day-30 death rate than non-ICU patients (odds ratio OR 2.12; 95 % confidence interval CI 1.66–2.72;
p
< 0.0001). The day-30 and early (<day 8) death rates increased over time in ICU (from 41.5 % the first to 56.9 % the last year of study (
p
= 0.001) and 28.7–38.8 % (
p
= 0.0292), respectively). Independent risk factors for day-30 death in ICU were age, arterial catheter,
Candida
species, preexposure to caspofungin, and lack of antifungal therapy at the time of blood cultures results (
p
< 0.05).
Conclusions
The availability of new antifungals and the publication of numerous guidelines did not prevent an increase of candidemia and death in ICU patients in the Paris area.
Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this ...study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.
Purpose
To assess the risk factors and outcomes associated with fungemia caused by the six most commonly occurring
Candida
species in patients with and without malignancies.
Methods
Analysis of the ...episodes of fungemia due to common
Candida
species in adults, based on an active hospital-based surveillance program (Paris area, France, 2002 to 2014).
Results
Of the 3417 patients (3666 isolates), 1164 (34.1%) had a solid tumor (45.7% digestive tract) and 586 (17.1%) a hematological malignancy (41.8% lymphoma, 33.5% acute leukemia). The hematology patients were significantly younger, more often pre-exposed to antifungals, more often infected by
C. tropicalis
,
C. krusei
, or
C. kefyr
, and more often treated in the first instance with an echinocandin. Compared with inpatients who were not in ICU at the time of fungemia, those in ICU were less frequently infected by
C. parapsilosis
(
p
< 0.02), had more recent surgery (
p
< 0.03), and died more frequently before day 8 and day 30 (
p
< 0.0001). An increase in crude mortality over time in ICU was observed only in oncology patients (
p
< 0.04). For all patients, lack of prescription of antifungals despite knowledge of positive blood culture increased the risk of death. The odds of being infected by a given
Candida
species compared with
C. albicans
were uneven regarding age, gender, type of malignancy, hospitalization in ICU, central venous catheter, HIV status, intravenous drug addiction, and previous exposure to antifungal drugs. Compared with
C. albicans
,
C. glabrata
(OR = 0.69 0.54–0.89) and
C. parapsilosis
(OR = 0.49 0.35–0.67) were associated with a decreased risk of death by day 8 and day 30.
Conclusion
The clinical context of underlying malignancy and hospitalization in ICU may be relevant to the initial management of candidemia.
Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal ...persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studied C. neoformans infection at the macrophage level (in vitro H99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing several C. neoformans populations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over time in vivo and in vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could exist in vitro and in vivo. C. neoformans exhibits a huge plasticity and adaptation to hosts that deserves further study. In vitro generation of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models.
Cryptococcus neoformans is a sugar-coated unicellular fungus that interacts closely with various cells and organisms, including amoebas, nematodes, and immune cells of mammals. This yeast is able to proliferate and survive in the intracellular environment. C. neoformans causes cryptococcosis, and yeast dormancy in humans has been suggested on the basis of epidemiological evidence obtained years ago. By studying an in vitro model of yeast-macrophage interaction and murine models of cryptococcosis, we observed that yeast cells evolve in heterogeneous populations during infection on the basis of global metabolic activity. We compared the growth ability and gene expression of yeast cells belonging to various populations in those two models. We eventually found a population of yeast cells with low metabolism that fit some of the criteria for dormant cells. This paves the way for further characterization of dormancy in C. neoformans.
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