Probing the role of point mutations in the cyp51A gene from Aspergillus fumigatus in the model yeast Saccharomyces cerevisiae

Azole-resistant strains of Aspergillus fumigatus have been detected and the underlying molecular mechanisms of resistance characterized. Point mutations in the cyp51A gene have been proved to be related to azole resistance in A. fumigatus clinical strains and with different resistance profiles depending on the amino acid change (G54E, G54V, G54R, G54W, M220V, M220K, M220T, M220I). The aim of this work was to express A. fumigatus cyp51A genes in the yeast Saccharomyces cerevisiae in order to better assess the contribution of each independent amino acid substitution to resistance. A tetracycline regulatable system allowing repression of the endogenous essential ERG11 gene was used. The expression of Aspergillus cyp51A alleles could efficiently restore the absence of ERG11 in S. cerevisiae. In general, S. cerevisiae clones expressing. A. fumigatus cyp51A alleles from azole-resistant isolates showed higher MICs to all azoles tested than those expressing alleles from susceptible isolates. The azole susceptibility profiles obtained in S. cerevisiae upon expression of specific cyp51A alleles recapitulated susceptibility profiles observed from their A. fumigatus origins. In conclusion this work supports the concept that characteristics of specific A. fumigatus cyp51A alleles could be investigated in the heterologous host S. cerevisia

Polymorphisms in host immunity modulating genes and risk of invasive aspergillosis: results from the aspBIOmics consortium

Recent studies suggest that immune-modulating single nucleotide polymorphisms (SNPs) influence the risk of developing cancer-related infections. Here, we evaluated whether 36 SNPs within 14 immune-related genes are associated with the risk of Invasive Aspergillosis (IA) and whether genotyping of these variants might improve disease risk prediction. We conducted a case-control association study of 781 immunocompromised patients, 149 of whom were diagnosed with IA. Association analysis showed that the IL4Rrs2107356 and IL8rs2227307 SNPs were associated with an increased risk of IA (OR=1.92, 95%CI: 1.20-3.09 and OR=1.73, 1.06-2.81) whereas the IL12Brs3212227 and IFN?rs2069705 variants were significantly associated with a decreased risk of developing the infection (OR=0.60, 0.38-0.96 and OR=0.63, 0.41-0.97). An allogeneic hematopoietic stem cell transplantation (allo-HSCT)-stratified analysis revealed that the effect observed for the IL4Rrs2107356 and IFN?rs2069705 SNPs was stronger in allo-HSCT (OR=5.63, 1.20-3.09 and OR=0.24, 0.10-0.59) than in non-HSCT patients, suggesting that the presence of these SNPs may render patients more vulnerable to infection especially under severe and prolonged immunosuppressive conditions. Importantly, in vitro studies revealed that carriers of the IFN?rs2069705C allele showed a significantly increased macrophage-mediated neutralisation of fungal conidia (P=0.0003) and, under stimulation conditions, produced higher levels of IFN? mRNA (P=0.049) and IFN? and TNFa cytokines (PLPS-96h=0.057, PPHA-96h=0.036 and PLPS+PHA-96h=0.030 and PPHA -72h=0.045, PLPS+PHA-72h=0.018, PLPS-96h=0.058 and PLPS+PHA -96h=0.0058, respectively). Finally, we also observed that the addition of SNPs significantly associated with IA to a model including clinical variables led to a substantial improvement in the discriminatory ability to predict the disease (AUC=0.659 vs. AUC=0.564, PLR=5.2•10-4 and P50.000Perm=9.34•10-5). These findings suggest that the IFN?rs2069705 SNP influences the risk of IA and that predictive models built with IFN?, IL8, IL12p70 and VEGFa variants might be used to predict disease risk and to implement risk-adapted prophylaxis or diagnostic strategies.This study was supported by grants PI12/02688 from the Fondo de Investigaciones Sanitarias (Madrid, Spain), PIM2010EPA-00756 from the ERA-NET PathoGenoMics (0315900A), and the Collaborative Research Center/Transregio 124 FungiNet. C.C. is supported by the Fundação para a Ciência e Tecnologia, Portugal (SFRH/BPD/96176/2013). This study also was supported by a donation of Consuelo González Moreno, an acute myeloid leukemia survivor. We thank Astella Pharma Inc. for supporting this work.info:eu-repo/semantics/publishedVersio

Statistical analyses of correlation between fluconazole MICs for Candida spp. assessed by standard methods set forth by the European Committee on Antimicrobial Susceptibility Testing (E.Dis. 7.1) and CLSI (M27-A2).

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) Subcommittee on Antifungal Susceptibility Testing recently published a standard for determining the susceptibility of fermentative yeasts to antifungals. From the beginning, the EUCAST and its North American counterpart, the CLSI, decided to work together in order to establish common standards. As part of this exercise, the susceptibility of a set of 475 yeast isolates was tested by both standards. The intraclass correlation coefficient and the equations defining the linear regression between both methods were estimated. Both methods produced very similar results, with an intraclass correlation coefficient of 0.954 (0.945 to 0.962), although linear regression analysis shows that the EUCAST standard resulted in slightly lower MICs. There were only eight isolates showing at least four twofold dilution MIC differences between both standards. After 24 h of incubation, the MICs obtained by the CLSI method were equivalent to those obtained by the EUCAST standard. In summary, both methods produce very similar MICs, indicating that methodology does not pose any obstacle to obtaining uniform standards for antifungal susceptibility testing of yeast

Susceptibility patterns and molecular identification of Trichosporon species

The physiological patterns, the sequence polymorphisms of the internal transcriber spacer (ITS), and intergenic spacer regions (IGS) of the rRNA genes and the antifungal susceptibility profile were evaluated for their ability to identify Trichosporon spp. and their specificity for the identification of 49 clinical isolates of Trichosporon spp. Morphological and biochemical methodologies were unable to differentiate among the Trichosporon species. ITS sequencing was also unable to differentiate several species. However, IGS1 sequencing unambiguously identified all Trichosporon isolates. Following the results of DNA-based identification, Trichosporon asahii was the species most frequently isolated from deep sites (15 of 25 strains; 60%). In the main, other Trichosporon species were recovered from cutaneous samples. The majority of T. asahii, T. faecale, and T. coremiiforme clinical isolates exhibited resistance in vitro to amphotericin B, with geometric mean (GM) MICs >4 mug/ml. The other species of Trichosporon did not show high MICs of amphotericin B, and GM MICs were <1 mug/ml. Azole agents were active in vitro against the majority of clinical strains. The most potent compound in vitro was voriconazole, with a GM MIC </=0.14 mug/ml. The sequencing of IGS correctly identified Trichosporon isolates; however, this technique is not available in many clinical laboratories, and strains should be dispatched to reference centers where these complex methods are available. Therefore, it seems to be more practical to perform antifungal susceptibility testing of all isolates belonging to Trichosporon spp., since correct identification could take several weeks, delaying the indication of an antifungal agent which exhibits activity against the infectious strain.S

Surveillance programs for detection and characterization of emergent pathogens and antimicrobial resistance: results from the Division of Infectious Diseases, UNIFESP

Several epidemiological changes have occurred in the pattern of nosocomial and community acquired infectious diseases during the past 25 years. Social and demographic changes possibly related to this phenomenon include a rapid population growth, the increase in urban migration and movement across international borders by tourists and immigrants, alterations in the habitats of animals and arthropods that transmit disease, as well as the raise of patients with impaired host defense abilities. Continuous surveillance programs of emergent pathogens and antimicrobial resistance are warranted for detecting in real time new pathogens, as well as to characterize molecular mechanisms of resistance. In order to become more effective, surveillance programs of emergent pathogens should be organized as a multicenter laboratory network connected to the main public and private infection control centers. Microbiological data should be integrated to guide therapy, adapting therapy to local ecology and resistance patterns. This paper presents an overview of data generated by the Division of Infectious Diseases, Federal University of São Paulo, along with its participation in different surveillance programs of nosocomial and community acquired infectious diseases.Várias alterações epidemiológicas ocorreram no perfil das doenças infecciosas hospitalares e comunitárias nos últimos 25 anos. Mudanças sociais e demográficas possivelmente relacionadas com esse fenômeno incluem o rápido crescimento populacional, o aumento da migração urbana e deslocamento através de fronteiras internacionais por turistas e imigrantes, alterações nos habitats de animais e artrópodes que transmitem doença assim como o aumento no número de pacientes com deficiências nas respostas de defesa. Os programas contínuos de vigilância de patógenos emergentes e resistência antimicrobiana são necessários para a detecção em tempo real de novos patógenos assim como para caracterizar mecanismos moleculares de resistência. Para serem mais efetivos, os programasde vigilância dos patógenos emergentes devem ser organizados em uma rede de laboratórios multicêntricos ligados aos principais centros de controle de infecções, públicos e privados. Os dados microbiológicos devem ser integrados a guias terapêuticos adaptando práticas terapêuticas à ecologia local eaos padrões de resistência. O artigo apresenta uma revisão dos dados gerados pela Disciplina de Infectologia, Universidade Federal de São Paulo (UNIFESP), contemplando sua participação nos diferentes programas de vigilância de doenças infecciosas hospitalares e adquiridas na comunidade.Universidade Federal de São Paulo (UNIFESP) Departamento de Medicina Divisão de Doenças InfecciosasUniversidade Federal de São Paulo (UNIFESP) Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, Depto. de Medicina Divisão de Doenças InfecciosasUNIFESP, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species

In recent years, triazole resistance in human Aspergillus diseases appears to have been increasing in several European countries. However, current data on the prevalence of resistance are based on a small number of studies which are only available from a few European countries. If present, triazole resistance can severely limit treatment options since alternatives, which are only available in intravenous form, have been shown to be associated with more side effects and poorer outcomes. Triazole resistance in Aspergillus spp. can evolve during therapy. Several point mutations, particularly in the cyp51A gene, have been associated with the development of resistance. Increasingly however, resistant isolates are also being detected in azole-naive patients. These isolates tend to have a particular genetic alteration consisting of a 34-base pair tandem repeat in the promoter coupled with a point mutation in the cyp51A target gene. This leads to an amino-acid substitution at codon 98 (TR34/L98H) causing multi-azole resistance. In patients whose Aspergillus isolates have developed resistance during azole therapy wildtype isolates, closely related genetically to the resistant isolates, have regularly been recovered from samples taken before the start of therapy or during an earlier phase. To date however, no isogenic isolate with a wild-type phenotype has been recovered from patients infected with an Aspergillus strain carrying the TR34/L98H genetic alteration. This suggests a possible environmental origin of the resistant fungus. This particular resistance mechanism has been observed most frequently in clinical isolates in the Netherlands where it has also been found in the environment. Moreover, the resistance mechanism has been demonstrated in clinical isolates in eight other European countries. Azole fungicides are widely used for crop protection and material preservation in Europe. They protect crops from disease, ensure yields and prevent fungal contamination of produce. It has been proposed that triazole resistance has evolved in the environment and could be driven by the selective pressure of azole fungicides. Although evidence supporting this hypothesis is growing, the link between the environmental use of azole fungicides and the development of triazole resistance in Aspergillus spp. is not yet proven. Triazole therapy has become the established treatment for invasive aspergillosis and is widely used in the treatment of allergic aspergillosis and chronic pulmonary aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis is usually prescribed for a minimum of 6–12 weeks, but often may need to be continued for months depending on the period of immunosuppression. Treatment of allergic aspergillosis and chronic pulmonary aspergillosis may need to continue for years or even throughout a patient’s lifetime. We estimated the burden of allergic, chronic and invasive aspergillosis using population statistics and published literature. Of the 733 million inhabitants in the European region1 [1], at any one time 2 100 000 patients may be suffering from allergic aspergillosis and 240 000 from chronic aspergillosis, that would be an indication for antifungal therapy. For invasive aspergillosis, we have estimated an annual incidence of 63 250 cases, complicating multiple underlying conditions including leukaemia, transplantation, chronic obstructive pulmonary disease (COPD) and medical intensive care. The inability to treat these patients with triazoles due to multi-azole resistance would have significant impact on patient management and associated health costs. Early and thorough investigation of this emerging public health problem is warranted in order to avoid the development and spread of resistance. This report examines current evidence for the environmental origin of resistance in Aspergillus spp. and makes recommendations for further steps to assess the risks and consequences of the environmental usage of azole derivatives. Improved surveillance of clinical isolates, including antifungal susceptibility testing, is the key to a better understanding of the magnitude of this emerging problem. Furthermore, the diagnosis of Aspergillus diseases needs to be improved and molecular methods allowing detection of resistance in culture-negative specimens must be further developed and implemented in laboratory practice. Finally, further environmental and laboratory studies are needed to confirm the environmental hypothesi

On the isoperimetric problem for the Laplacian with Robin and Wentzell boundary conditions

Doctor of PhilosophyWe consider the problem of minimising the eigenvalues of the Laplacian with Robin boundary conditions $\frac{\partial u}{\partial \nu} + \alpha u = 0$ and generalised Wentzell boundary conditions $\Delta u + \beta \frac{\partial u}{\partial \nu} + \gamma u = 0$ with respect to the domain $\Omega \subset \mathbb R^N$ on which the problem is defined. For the Robin problem, when $\alpha > 0$ we extend the Faber-Krahn inequality of Daners [Math. Ann. 335 (2006), 767--785], which states that the ball minimises the first eigenvalue, to prove that the minimiser is unique amongst domains of class $C^2$. The method of proof uses a functional of the level sets to estimate the first eigenvalue from below, together with a rearrangement of the ball's eigenfunction onto the domain $\Omega$ and the usual isoperimetric inequality. We then prove that the second eigenvalue attains its minimum only on the disjoint union of two equal balls, and set the proof up so it works for the Robin $p$-Laplacian. For the higher eigenvalues, we show that it is in general impossible for a minimiser to exist independently of $\alpha > 0$. When $\alpha < 0$, we prove that every eigenvalue behaves like $-\alpha^2$ as $\alpha \to -\infty$, provided only that $\Omega$ is bounded with $C^1$ boundary. This generalises a result of Lou and Zhu [Pacific J. Math. 214 (2004), 323--334] for the first eigenvalue. For the Wentzell problem, we (re-)prove general operator properties, including for the less-studied case $\beta 0$ establish a type of equivalence property between the Wentzell and Robin minimisers for all eigenvalues. This yields a minimiser of the second Wentzell eigenvalue. We also prove a Cheeger-type inequality for the first eigenvalue in this case

Resolution of disseminated fusariosis in a child with acute leukemia treated with combined antifungal therapy: a case report

<p>Abstract</p> <p>Background</p> <p><it>Fusarium </it>spp. is being isolated with increasing frequency as a pathogen in oncohematologic patients. Caspofungin and amphotericin B have been reported to have synergistic activity against <it>Fusarium </it>spp.</p> <p>Case presentation</p> <p>We herein report a case of disseminated fusariosis diagnosed by chest CT scan and positive blood cultures to <it>Fusarium </it>spp. Because the patient's clinical condition deteriorated, CRP levels increased, and blood cultures continued to yield <it>Fusarium </it>spp. despite liposomal amphotericin B monotherapy up to 5 mg/kg daily, treatment with caspofungin was added. Within 2 weeks of onset of combined antifungal therapy, the chest CT scan demonstrated a progressive resolution of the pulmonary lesions. Upon discontinuation of intravenous antifungals, the patient received suppressive therapy with oral voriconazole. Three months later, a chest CT scan showed no abnormalities. Twenty-five months after discontinuation of all antifungal therapy, the patient remains in complete remission of her neoplastic disease with no signs of clinical activity of the <it>Fusarium </it>infection.</p> <p>Conclusion</p> <p>This is the first description of successful treatment of disseminated fusariosis in a pediatric patient with acute lymphoblastic leukemia with caspofungin and amphotericin B followed by oral suppressive therapy with voriconazole.</p

Contribution of Candida biomarkers and DNA detection for the diagnosis of invasive candidiasis in ICU patients with severe abdominal conditions

BACKGROUND: To assess the performance of Candida albicans germ tube antibody (CAGTA), (1 → 3)-ß-D-glucan (BDG), mannan antigen (mannan-Ag), anti-mannan antibodies (mannan-Ab), and Candida DNA for diagnosing invasive candidiasis (IC) in ICU patients with severe abdominal conditions (SAC). METHODS: A prospective study of 233 non-neutropenic patients with SAC on ICU admission and expected stay ≥ 7 days. CAGTA (cutoff positivity ≥ 1/160), BDG (≥80, 100 and 200 pg/mL), mannan-Ag (≥60 pg/mL), mannan-Ab (≥10 UA/mL) were measured twice a week, and Candida DNA only in patients treated with systemic antifungals. IC diagnosis required positivities of two biomarkers in a single sample or positivities of any biomarker in two consecutive samples. Patients were classified as neither colonized nor infected (n = 48), Candida spp. colonization (n = 154) (low-grade, n = 130; high-grade, n = 24), and IC (n = 31) (intra-abdominal candidiasis, n = 20; candidemia, n = 11). RESULTS: The combination of CAGTA and BDG positivities in a single sample or at least one of the two biomarkers positive in two consecutive samples showed 90.3 % (95 % CI 74.2–98.0) sensitivity, 42.1 % (95 % CI 35.2–98.8) specificity, and 96.6 % (95 % CI 90.5–98.8) negative predictive value. BDG positivities in two consecutive samples had 76.7 % (95 % CI 57.7–90.1) sensitivity and 57.2 % (95 % CI 49.9–64.3) specificity. Mannan-Ag, mannan-Ab, and Candida DNA individually or combined showed a low discriminating capacity. CONCLUSIONS: Positive Candida albicans germ tube antibody and (1 → 3)-ß-D-glucan in a single blood sample or (1 → 3)-ß-D-glucan positivity in two consecutive blood samples allowed discriminating invasive candidiasis from Candida spp. colonization in critically ill patients with severe abdominal conditions. These findings may be helpful to tailor empirical antifungal therapy in this patient population