Ecology of <i>Cryptococcus neoformans</i> in Vietnam

Cryptococcal meningitis is an infection with Cryptococcus species; almost all disease occurs in immunosuppressed patients due to C. neoformans. Cryptococcus spp are environmental saprophytes; the ability to cause disease is considered an accident of adaptation to their ecological niche. In Southeast Asia, cryptococcal meningitis in immunocompetent individuals is well-described and is almost always due to the C. neoformans ST5 (VNIa-5) lineage. I hypothesize this lineage has relative increased pathogenicity. The aims of this thesis were to: identify the ecological niche of C. neoformans; define the diversity of C. neoformans in Vietnam; compare the virulence of different genotypes, and; define transcriptional differences associated with virulence. I identified spatial clustering of lineages VNIa-4, VNIa-5 and VNIa-32. Environmental sampling yielded 123 cryptococci the majority of which were non-pathogenic. Using the G. mellonella infection model I identified an ‘ecological imprint’ on isolates. Within a lineage, the virulence of an isolate is determined by its source – environmental, immunosuppressed or immunocompetent patient. Moreover, the virulence of environmental C. neoformans can be upregulated by clinical strains of that lineage. This ‘induction’ effect is regulated by a protein/peptide signaling system. Comparative transcriptomics revealed 1700 genes differentially expressed between clinical and environmental isolates. Gene ontology (GO) terms relevant to metabolism and cellular biosynthesis were enriched in clinical isolates; GO terms relevant to cell cycle and cell division were significantly enriched in induced environmental isolates. Clinical and induced environmental isolates had highly similar transcriptomes. A number of genes previously confirmed as virulence determinants were upregulated in clinical and induced environmental isolates; a third of differentially expressed genes between the induced and `naive' isolates were hypothetical proteins. These are novel candidates implicated in the upregulation of virulence. My findings provide insight into ecology of C. neoformans in Vietnam and the genetic control of virulence

Enhancement of flow boiling heat transfer in microchannels by nano- and micro-surface treatments

This work investigates the flow boiling heat transfer in microchannels with the aim of developing compact cooling systems which can be adapted to miniaturized power components. Nano and micro-surface treatments were used as innovative techniques to improve the heat transfer performance as well as to delay the intermittent dryout. It was observed that the micro-structured surfaces show significant enhancements (up to 85%) in heat transfer compared to the smooth surfaces. Especially, using the highly-wetted structured surface, the intermittent dryout is improved

Multilocus sequence typing of Cryptococcus neoformans var. grubii from Laos in a regional and global context.

Cryptococcosis causes approximately 180 000 deaths each year in patients with human immunodeficiency virus (HIV). Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii, responsible for the majority of cases, is distributed globally. We used the consensus ISHAM Multilocus sequence typing (MLST) scheme to define the population structure of clinical C. neoformans var. grubii isolates from Laos (n = 81), which we placed into the global context using published MLST data from other countries (total N = 1047), including a reanalysis of 136 Vietnamese isolates previously reported. We observed a phylogeographical relationship in which the Laotian population was similar to its neighbor Thailand, being dominated (83%) by Sequence Types (ST) 4 and 6. This phylogeographical structure changed moving eastwards, with Vietnam's population consisting of an admixture of isolates dominated by the ST4/ST6 (35%) and ST5 (48%) lineages. The ST5 lineage is the predominant ST reported from China and East Asia, where it accounts for >90% of isolates. Analysis of genetic distance (Fst) between different populations of C. neoformans var. grubii supports this intermediate structure of the Vietnamese population. The pathogen and host diversity reported from Vietnam provide the strongest epidemiological evidence of the association between ST5 and HIV-uninfected patients. Regional anthropological genetic distances suggest diversity in the C. neoformans var. grubii population across Southeast Asia is driven by ecological rather than human host factors. Where the ST5 lineage is present, disease in HIV-uninfected patients is to be expected

Assessing the virulence of Cryptococcus neoformans causing meningitis in HIV infected and uninfected patients in Vietnam.

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in individuals who are uninfected with human immunodeficiency virus (HIV). This disease was associated with a single genotype of Cryptococcus neoformans (sequence type [ST]5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans, we selected 30 representative Vietnamese isolates and compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37°C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (P < .001). Counterintuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF-α concentrations (P < .001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts

Comparative genomics of Cryptococcus neoformans var. grubii associated with meningitis in HIV infected and uninfected patients in Vietnam.

The vast burden of cryptococcal meningitis occurs in immunosuppressed patients, driven by HIV, and is caused by Cryptococcus neoformans var. grubii. We previously reported cryptococcal meningitis in Vietnam arising atypically in HIV uninfected, apparently immunocompetent patients, caused by a single amplified fragment length polymorphism (AFLP) cluster of C. neoformans var. grubii (VNIγ). This variant was less common in HIV infected individuals; it remains unclear why this lineage is associated with apparently immunocompetent patients. To study this host tropism we aimed to further our understanding of clinical phenotype and genomic variation within Vietnamese C. neoformans var. grubii. After performing MLST on C. neoformans clinical isolates we identified 14 sequence types (STs); ST5 correlated with the VNIγ cluster. We next compared clinical phenotype by lineage and found HIV infected patients with cryptococcal meningitis caused by ST5 organisms were significantly more likely to have lymphadenopathy (11% vs. 4%, p = 0.05 Fisher's exact test) and higher blood lymphocyte count (median 0.76 versus 0.55 X109 cells/L, p = 0.001, Kruskal-Wallis test). Furthermore, survivors of ST5 infections had evidence of worse disability outcomes at 70 days (72.7% (40/55) in ST5 infections versus 57.1% (52/91) non-ST5 infections (OR 2.11, 95%CI 1.01 to 4.41), p = 0.046). To further investigate the relationship between strain and disease phenotype we performed genome sequencing on eight Vietnamese C. neoformans var. grubii. Eight genome assemblies exhibited >99% nucleotide sequence identity and we identified 165 kbp of lineage specific to Vietnamese isolates. ST5 genomes harbored several strain specific regions, incorporating 19 annotated coding sequences and eight hypothetical proteins. These regions included a phenolic acid decarboxylase, a DEAD-box ATP-dependent RNA helicase 26, oxoprolinases, a taurine catabolism dioxygenase, a zinc finger protein, membrane transport proteins and various drug transporters. Our work outlines the complexity of genomic pathogenicity in cryptococcal infections and identifies a number of gene candidates that may aid the disaggregation of the pathways associated with the pathogenesis of Cryptococcus neoformans var. grubii

An open label randomized controlled trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis

Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anti-cancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods:Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first two weeks - or standard care plus tamoxifen 300mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031 . Results: 50 patients were enrolled, (median age 34 years, 35 male). Tamoxifen had no effect on EFA (- 0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference - 0.005log10CFU/ml/day, 95%CI: -0.16, 0.15, P=0.95). Tamoxifen caused QTc prolongation. Conclusion: High dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding:The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA

Approche microscopique de la génération de chaleur par frottement et effet Joule

Dans le cadre de l'étude des phénomènes de transfert de chaleur, nous désirons répondre aux problématiques liées à la génération de chaleur par frottement sur des dispositifs de dimensions submicrométriques. Notre étude a été réalisée à l'aide d'un microscope à effet de force atomique (AFM : Atomic Force Microscope) instrumenté d'une sonde thermique en contact sur un échantillon plan. Le premier objectif est de retrouver les flux de chaleur par frottement générés vers la sonde et vers l'échantillon lorsque la sonde se déplace sur la surface de l'échantillon. Le deuxième objectif est de quantifier la chaleur générée par effet Joule au contact entre la sonde et un échantillon conducteur. Nous avons d'abord établi des modèles analytiques permettant de comprendre les relations entre les flux de chaleur et les variations des températures de la sonde et de l'échantillon. Ensuite, nous avons simulé ces modèles pour calculer les coefficients de sensibilité de ces deux dispositifs dans le but de savoir si la détection de leurs variations de température par frottement sont possibles. Nous avons conçu un procédé pour retrouver expérimentalement les coefficients de sensibilité de la sonde. La compatibilité entre les valeurs expérimentales et les valeurs simulées valident nos modèles et les mesures. Après, nous avons réalisé des mesures de variation de température de la sonde lorsque celle-ci balaye l'échantillon. Grâce à ces études, nous avons proposé une hypothèse sur les caractéristiques du flux de chaleur par frottement à l'échelle microscopique. Enfin, nous avons conçu une expérience pour mesurer la résistance électrique de contact entre la sonde et un échantillon en cuivre

EFFETS DES NANO- ET MICRO-STRUCTURATIONS DE SURFACE SUR LE TRANSFERT THERMIQUE EN ÉBULLITION

Septembre 2010This work investigates the flow boiling heat transfer in microchannels with the aim of developing compact cooling systems which can be adapted to miniaturized power components. Nano and micro-surface treatments were used as innovative techniques to improve the heat transfer performance as well as to delay the intermittent dryout. Initially, pool-boiling experiments were performed to highlight the impact of nanocoatings on nucleate-boiling mechanisms. It was observed that the surface wettability modified by nanoparticle deposition had significant effects on the boiling processes. Afterwards, a second experimental campaign was conducted to investigate the flow boiling in a microchannel with nanocoated and microstructured samples. These studies highlighted the impacts of surface wettability and of micro-patterning on two-phase flow patterns, pressure drop and heat transfer coefficient. In particular, significant enhancements in heat transfer coefficient and in intermittent dryout were obtained with micro-structured samples.Ce travail concerne l'étude du transfert thermique par ébullition dans des microcanaux avec l'objectif de développer des systèmes thermiques compacts adaptés aux composants de puissance miniaturisés. Les techniques de nano- et micro-structurations de surface ont été utilisées comme des méthodes novatrices pour améliorer les performances de transfert thermique ainsi que pour retarder l'assèchement intermittent qui apparaît dans les microcanaux. Dans un premier temps, des expériences d'ébullition en vase ont été réalisées afin de mettre en évidence l'impact d'un revêtement à l'échelle nanométrique sur les mécanismes d'ébullition nucléée. Il a été observé que la mouillabilité de surface, qui est modifiée par le dépôt de nanoparticules, a des effets significatifs sur les processus d'ébullition. Dans un deuxième temps, une campagne expérimentale a été réalisée pour étudier l'ébullition convective dans un microcanal avec des échantillons nano- et microstructurées. Ces études ont mis en évidence les effets de la mouillabilité et de la microstructuration de surface sur les régimes d'écoulement, les pertes de charge et le coefficient de transfert thermique. En particulier, de fortes améliorations du coefficient de transfert thermique et le retard d'assèchement intermittent ont été obtenus avec des surfaces microstructurées