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While large outbreaks of C. auris in hospital settings have been described, few clear cases of direct transmission have been documented. We here investigated the transmission of C. auris clade I between two patients with a 41- to 61-day delay between exposure and the development of colonization. This may lead to changes in the recommendations concerning treatment of C. auris cases, as an incubation period of this length is one of the first to be reported.

Cryptococcal antigen (CrAg) is a capsule polysaccharide antigen that can be detected in the fluids of patients with cryptococcal infections. Cryptococcal Antigen Latex Agglutination System (CALAS), enzyme-linked immunosorbent assays (EIA), and lateral flow assay (LFA) are the main methods available. Two main commercial LFA kits are available: CryptoPS (Biosynex, Illkirch Graffenstaden, France) and CrAg LFA (IMMY, Inc. USA). In our lab, we prospectively used CryptoPS as a screening tool in serum for confirmed positive results with CALAS. We investigated the rigor of the CryptoPS test in serum in a multicentric evaluation over 3 years. To improve the specificity of CryptoPS in serum, we additionally implemented and evaluated a pretreatment protocol before CryptoPS testing. A total of 43 serum samples collected from 43 patients were investigated. We found that the CryptoPS assay is hampered by a high rate of false-positive results in serum with a high rate of CryptoPS-positive but CrAg LFA-negative and CALAS-negative sera in patients with no proof of Cryptococcus infection (n = 29). Using a simple pretreatment procedure (5 min incubation at 100°C and centrifugation) we were able to reverse false-positive results, suggesting that there could be interferent material present in the serum. Pretreatment also impacted the CryptoPS results (negative result) in two patients with the cryptococcal disease, one with isolated antigenemia and one with cryptococcal meningitis. Comparing the titers obtained with CALAS and CrAg LFA, we noticed that the titer obtained with CrAg LFA was almost 10-fold higher than those with CALAS. This study showed that Biosynex CryptoPS in serum could give false-positive results even in the absence of cryptococcal disease. These could be reduced by applying an easy pretreatment procedure to the serum before testing, with little but existing impact on the sensitivity.Lateral flow assays are useful to detect the cryptococcal antigen in human fluids. We investigated CryptoPS-positive results and observed that true false-positive results occurred. The false-positive results can be reduced by applying an easy pretreatment procedure.

S1.4 Fungal infections in Asia, bringing it out of the dark, September 21, 2022, 11:00 AM - 12:30 PM Background Routine laboratory testing for cryptococcal meningitis currently consists of Cryptococcal antigen (CrAg) testing in blood and cerebrospinal fluid (CSF), CSF India ink, and CSF fungal culture. Quantitative cryptococcal culture (QCC) is labor intensive and not feasible in most settings. Objectives We evaluated quantitative (qPCR) and reverse transcriptase qPCR (RT-qPCR) assays to quantify cryptococcal load in CSF, plasma, and blood. We also investigated the dynamics of fungal DNA and RNA detection during antifungal treatment. Methods We developed a qPCR assay that can differentiate serotypes A, D, and B/C of Cryptococcus neoformans and C. gattii based on the amplification of a unique nuclear Quorum sensing protein 1 (QSP1) and a multicopy 28S rRNA gene and evaluated the assays on 205-patient samples from the AMBITION-cm trial in Botswana and Malawi (2018-2021). CSF, plasma, and whole blood samples were stored per patient and were sampled at day 0 (baseline), day 7 and 14 for CSF and at day 1, 3 and 7 for plasma and whole blood post antifungal treatment initiation. A Roche LightCycler480 and Graph pad prism were used for data analysis. Results A total of 205/209 stored patient samples (85 from Botswana; 124 from Malawi), were used. For QSP1 qPCR tested in CSF at D0, 138 (81.7%) were serotype A, 28 (16.6%) were serotype B/C and 3 (1.8%) were a mixed infection of serotype A and B/C. There was no amplification with 36 (17.6%) samples. There was no difference in fungal loads at D0, D7, and D14 between serotype A and B/C with the QSP1 qPCR assay, and QCC. QCC showed a good correlation with qPCR quantification with QSP1 qPCR (slope = 0.797, R2 = 0.73) and with 28S rRNA qPCR (Slope = 0.771, R2 = 0.778) assays. The fungal load at D0 was significantly higher in patients who died at week 2 (w2) and at week 10 (w10) as compared with patients who survived post-week 10 (P .05). Detection of Cryptococcus DNA (28S rRNA qPCR) in plasma or whole blood within the first 24 h of treatment was significantly associated with early mortality at w2 and mortality at w10 (P Conclusion Quantification of C. neoformans and C. gattii load in CSF and plasma at D0 is useful in identifying patients at risk of death and may be a promising tool for monitoring treatment response in the future.

International audience; It is becoming increasingly obvious that glycophosphatidylinositol (GPI)-anchored proteins (GAPs) play a prominent role in fungi, a full understanding of GAPs is however lacking especially for the human opportunistic fungus Cryptococcus neoformans. Using online GPI prediction tools, GAPs were identified and subsequently a mutant library for these GAP-encoding genes was developed and a publicly available knock out (KO) mutant library was used. In total, 41 overexpression and 34 KO mutants, representing 47 unique genes, were analyzed. From the analysis of the two libraries, two main gene candidates, a mannoprotein 88 (MP88) (CNAG_00776) and an uncharacterized protein (CNAG_00137) were further investigated by constructing additional independent mutant strains. The CNAG_00776 mutant showed an impaired growth upon plasma membrane stress and significant decreased phagocytosis. The CNAG_00137 mutant showed impaired growth during cell wall stress or increased temperature as well as decreased phagocytosis compared. By performing a large genetic screen of GAPs in the genome of the human fungal pathogen C. neoformans, we identified two candidate GAP genes involved in C. neoformans/host interaction and stress response. Further research into these two genes could potentially result in new targets for antifungals, treatment strategies or vaccines to manage C. neoformans disease.

Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H. capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

International audience; Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30–40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast., Author summary Quiescence/dormancy in microorganism is a common feature that enables survival at the population level. In fungi, quiescence has been studied in the baker yeast Saccharomyces cerevisiae. In Cryptococcus neoformans, dormancy is of great interest since it is known from the natural history of cryptococcosis that dormancy in yeast can last decades exists before possible reactivation upon immunosuppression. Based on a previous study which identified a subpopulation of dormant yeasts in experimental models of cryptococcosis, we identified here in vitro conditions that enabled the induction of dormancy via the formation of viable but non culturable cells (VBNC). Reactivation of part of these cells was possible through stimulation with vitamin B5, a quorum sensing molecule. We showed that the global metabolism of the VBNC was down but harbored specific signatures compared to control conditions. We identified mitochondrial metabolism, in particular the fatty acid pathway, as key for the maintenance and viability of VNBC. These findings open the road for research on dormancy. Elucidating the parameters involved will help understand the pathophysiology of the disease including the difficulty in eradication of the yeasts despite therapy, and the possible relapse/recurrence of the infection.

A remarkable aspect of the human fungal pathogen Cryptococcus neoformans is the morphological changes triggered by the interaction with the host. During infection, a specific morphological change in cell size is observed, particularly in lung tissue, from regular 5 to 7 µm cells (RCs) to titan cells (TCs, > 10 µm and up to 100 µm). However, the stable and reproducible generation of large quantity of TCs was only possible during experimental infection. We implemented here in vitro conditions allowing TCs generation with the aim to understand the ancestry of TC, the environmental determinants of TCs formation and perform easily phenotype/genotype analysis to uncover genetic factors underlying TCs formation. This paper reports robust in vitro conditions that generates yeasts cells harboring the main characteristics of TCs: large (>10 µm), uninucleate and polyploid cells harboring a single large vacuole with a dense capsule surrounding a thickened melanized cell wall. We observed that TCs formation begins as soon as 8 hours after induction, with a maximal proportion of TCs obtained after 24 hours. TCs derived for the initial oldest cells (mother cells) rather than from daughter cells. Hypoxia, nutrient starvation and low pH stresses were the main factors that trigger TCs formation, while quorum sensing factors like the initial inoculum concentration and the addition of pantothenic acid also impacted TCs formation. Antifungal drugs, resulting in inhibition of ergosterol (fluconazole), or proteins and nucleic acids (flucytosine), altered TCs formation, as did hosts-related products such as serum, phospholipids and anti-capsular antibodies in our settings. We then explored genetic factors important for TCs formation using two approaches. Using strains from H99 lineage strains among which few genetic differences have been described, we showed that TCs formation was dependent on Lmp1, Sgf29 and Gpr4/5-Rim101 proteins. Then, based on a the analysis of natural Pkr1 loss-of-function clinical isolates and serial clinical isolates, we observed the important role of Pkr1 protein, a negative regulator of the cyclic adenosine monophosphate / protein kinase A (cAMP/PKA), for TCs formation. These strains also emphasize the structural and functional relationship between Pkr1 and Pka. Our results provide new insights into TCs biogenesis with identification of multiple important factors/pathways involved. The implementation of such standardized and robust in vitro conditions pave the way for future research focusing on the genetic basis of TCs biogenesis, biology of TCs and the ontology of morphological changes in Cryptococcus neoformans. Author Summary Cryptococcus neoformans is a yeast that is capable of morphological change upon interaction with host. Particularly, in the lung of infected mice, a subpopulation of yeast evolves toward gigantism (titan cells size from 10 to 100 µm) that include other characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of large number of such cells outside the lung of mice have been described but was not reproducible nor standardized. We here report standardized, reproducible, robust conditions of generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that Titan cells were generated upon stresses such as change in the medium, nutrient deprivation, hypoxia and low pH. Using collection of well characterized reference strains and clinical isolates, we validated with our model the AMPc/PKA/Rim pathway as the major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes in Cryptococcus neoformans.

International audience; Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergil-losis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to LAmB (50 to 400 μg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200 μg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proin-flammatory cytokines was quantified at 6 and 24 h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with LAmB or empty liposomes, even at 400 μg/ml. Only the highest concentration tested of LAmB (400 μg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50 μg/ ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6 h with 50 μg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after LAmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.

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). Sí

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., IMPORTANCE 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.

Introduction Des etudes experimentales et cliniques ont suggere l’interet de l’administration d’aerosols d’amphotericine B pour la prophylaxie de l’aspergillose invasive. La moindre toxicite cellulaire de l’amphotericine B liposomale (L-AmB) par rapport a l’amphotericine B desoxycholate (D-AmB) a ete montree par des tests colorimetriques de viabilite cellulaire en point final. Cependant, ces tests ne peuvent pas mesurer la viabilite de cellules adherentes en culture cellulaire de facon non invasive et en temps reel. Notre objectif etait donc de suivre la viabilite cellulaire en temps reel sur une lignee de cellules epitheliales alveolaires en utilisant une technologie basee sur l’impedance cellulaire et d’etudier le niveau d’expression de genes de cytokines pro-inflammatoires apres exposition a L-AmB ou D-AmB. Methodes Des cellules epitheliales alveolaires A549 ont ete cultivees dans des puits contenant des electrodes (plaques xCELLigence, ACEA Biosciences) permettant des mesures d’impedance en continu. Les resultats sont exprimes en index cellulaires (IC) mesures sur une periode de 100 h, prenant en compte l’adhesion cellulaire aux electrodes et globalisant divers etats biologiques comme la proliferation, la viabilite et la morphologie. Les cellules A549 ont ete ensemencees a une concentration de 18000 cellules/puits et, apres 23 h de culture, les antifongiques (50 a 400 μg/ml de D-AmB ou L-AmB) ou des concentrations equivalentes de liposomes vides ont ete ajoutes en quadruplicat. En parallele, 2 plaques de culture ont ete utilisees pour quantifier l’expression des genes de 2 cytokines pro-inflammatoires (TNF-α et IL-8) 6 h et 24 h apres l’addition des drogues par RT-PCR en temps reel sur les lysats cellulaires. Resultats Une diminution de l’IC a ete observee avec le D-AmB a partir d’une concentration de 50 μg/ml avec une recuperation des cellules a 60 h. Avec des concentrations plus elevees de D-AmB, aucune recuperation cellulaire n’a ete observee. Au contraire, aucune alteration de l’IC n’a ete observee avec le L-AmB ou avec les liposomes vides, meme a 400 μg/ml. Aucune augmentation de l’expression de genes de cytokines n’a ete observee a 6 h, ni avec les liposomes vides, ni avec le L-AmB excepte une induction de 2 fois et de 6 fois de l’ARNm du TNF-α a 200 et 400 μg/ml, respectivement, et une induction de 4 fois de l’ARNm de l’IL-8 a 400 μg/ml. Au contraire, meme avec une faible concentration de D-AmB (50 μg/ml), des inductions de 4 fois et 7 fois du TNF-α et de l’IL-8, respectivement, ont ete observees a 6 h. Conclusions La mesure de l’impedance cellulaire en continu est un outil interessant pour suivre la toxicite cellulaire. La cinetique en temps reel de l’impedance cellulaire et la mesure de l’expression de genes de cytokines pro-inflammatoires ont confirme la meilleure tolerance cellulaire de l’amphotericine B liposomale compare a l’amphotericine B desoxycholate.

Introduction La PCR digitale permet une quantification absolue des acides nucleiques de facon independante de l’efficacite de PCR. Son principe est de repartir l’ADN dans plusieurs milliers gouttelettes (10°000 a 20°000) realisant autant de PCR independantes. La detection de la reaction de PCR se fait en point final, base sur la detection d’une fluorescence. Les resultats s’analysent en comptant le nombre de gouttelettes positives par rapport a leur nombre total et permet ensuite de calculer un nombre de copie/μL d’ADN detecte. Dans le cas des cibles repetees et particulierement celles repetees en tandem, il est d’usage d’utiliser une digestion enzymatique de l’ADN pour liberer chaque copie de leurs voisines et eviter que deux copies ne soient presentes dans une gouttelette. Le nombre de copie estimee de l’ADN ribosomal 28S (ADNrib) a ete estime a 35 lors du sequencage d’Aspergillus fumigatus. Objectif L’objectif de l’etude etait de quantifier le nombre de copies d’ADNrib (28S) de differents isolats d’Aspergillus fumigatus en comparaison a un gene unique du genome (FKS1) et de comparer l’effet de la digestion enzymatique sur les ADN de ces souches a celui de la digestion d’ADNrib circulant issus de serums de patients avec aspergillose invasive. Methodes La souche de reference AF293 (souche sequencee en 2005) et 9 isolats cliniques de genotypes et de groupes genetiques differents ont ete selectionnes et extraits de maniere standardisee (MagNapure, Roche). Le serum de dix patients atteints d’aspergillose invasive ont ete selectionnes. Chaque ADN a subit une digestion enzymatique (EcoRI et HaeIII apres avoir verifier l’absence de site de restriction dans la region amplifiee) et compare au meme ADN sans digestion. Les PCR 28S de Challier et al. [1] et FKS1 de Herrera et al. [2] ont ete utilisee en duplex. Les resultats de digitale ont ete compares a la PCR quantitative classique. Resultats Le nombre de copie de 28S variait de 61 a 86 copies (mediane : 75) en fonction des souches cliniques. L’ADN d’Af293 evaluee a 35 copies a ete retrouve dans nos experiences a 69 ± 7 copies. En absence de traitement enzymatique, le nombre de copies etait significativement plus bas et variait de 34 a 76 copies (mediane : 57) (p = 0,003). A l’inverse, aucun impact significatif du traitement enzymatique n’a ete observe pour les 10 ADN circulants avec une mediane de 0,5 copies/μL sans digestion versus 0,85 copies/μL d’ADNrib avec digestion (p = 0,19). Ces differences ont ete observees dans les memes proportions en qPCR. Conclusion Ces donnees generees en digital PCR permettent de mettre en evidence que l’ADN circulant au cours de l’aspergillose invasive serait de moins grande taille que l’ADN extrait de souches, renforcant l’hypothese que l’ADN aspergillaire est circulant et libre.

International audience; Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

International audience; In the present report, we revisited the B cell responsiveness of 7 wild-derived mouse strains to various toll-like receptor ligands (TLR-L). We found that 2 of them, namely PWK and STF presented profound defects in B cell proliferative responses to most of the TLR-L. Yet, their macrophage responses were largely unaffected, suggesting that regulation of TLR pathways are distinct in B cells and macrophages. We also showed that, anti-CD40 mAbs rescued the low proliferative responses to CpG in both PWK and STF B cells. In the other hand, CpG synergized with LPS to induce high levels of proliferation in STF B cells, which did not respond to LPS alone. Cytokine or immunoglobulin (Ig) productions, in vitro, were less impaired than the proliferative responses to LPS or CpG alone. In STF B cells, both ERK, P38 and JNK pathways were affected following in vitro TLR4 or TLR9 signaling. Moreover, while the basal levels of Ig secreting cells and of serum Igs were similar to that of control mice, antibody responses to both TI and TD antigens were severely affected, mainly in STF mice. Our findings therefore highlight the relevance of wild-derived mouse strains and TLR-L to study B cell physiology.

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal qPCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicentre and monocentre evaluation of PCP qPCR assays was performed. For the multicentre study, 16 reference laboratories from eight different countries, performing 20 assays analysed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads respectively). The monocentre study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, Reverse Transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation