Comparative assessment of immune evasion mechanisms in human whole-blood infection assays by a systems biology approach.

Computer simulations of mathematical models open up the possibility of assessing hypotheses generated by experiments on pathogen immune evasion in human whole-blood infection assays. We apply an interdisciplinary systems biology approach in which virtual infection models implemented for the dissection of specific immune mechanisms are combined with experimental studies to validate or falsify the respective hypotheses. Focusing on the assessment of mechanisms that enable pathogens to evade the immune response in the early time course of a whole-blood infection, the least-square error (LSE) as a measure for the quantitative agreement between the theoretical and experimental kinetics is combined with the Akaike information criterion (AIC) as a measure for the model quality depending on its complexity. In particular, we compare mathematical models with three different types of pathogen immune evasion as well as all their combinations: (i) spontaneous immune evasion, (ii) evasion mediated by immune cells, and (iii) pre-existence of an immune-evasive pathogen subpopulation. For example, by testing theoretical predictions in subsequent imaging experiments, we demonstrate that the simple hypothesis of having a subpopulation of pre-existing immune-evasive pathogens can be ruled out. Furthermore, in this study we extend our previous whole-blood infection assays for the two fungal pathogens Candida albicans and C. glabrata by the bacterial pathogen Staphylococcus aureus and calibrated the model predictions to the time-resolved experimental data for each pathogen. Our quantitative assessment generally reveals that models with a lower number of parameters are not only scored with better AIC values, but also exhibit lower values for the LSE. Furthermore, we describe in detail model-specific and pathogen-specific patterns in the kinetics of cell populations that may be measured in future experiments to distinguish and pinpoint the underlying immune mechanisms.

[Etiological significance and antibioticosensetivity of certain microorganisms in aggravation of chronical bronchopulmonary pathology in workers of diverse economic sectors.]

It has been shown that in patients with upper respiratory diseases of occupational etiology gram negative flora prevail (38% of cases). They are followed by yeast-like fungi (up to 36% of cases), gram positive flora - 26%. The most effective antibacterial agents for treating golden staphilococcus in patients of the group studied are cefotaxime, sparfloxacine, levofloloxacine. Cefotoxime, ceftriaxon, ciprofloxacine are used against intestinal bacteria. Cefepim, ceftazidim are used against non-fermenting gram negative bacteria. C.Albicans can be treated with amfotericine and fluconazol.

Overcoming the Resistance Hurdle: Pharmacokinetic-Pharmacodynamic Target Attainment Analyses for Rezafungin (CD101) against Candida albicans and Candida glabrata.

Rezafungin (CD101) is a novel echinocandin antifungal agent with activity against Aspergillus and Candida species, including azole- and echinocandin-resistant isolates. The objective of these analyses was to conduct pharmacokinetic (PK)-pharmacodynamic (PD) target attainment analyses to evaluate single and once-weekly rezafungin dosing to provide dose selection support for future clinical studies. Using a previously developed rezafungin population PK model, Monte Carlo simulations were conducted utilizing the following three intravenous rezafungin regimens: (i) a single 400 mg dose, (ii) 400 mg for week 1 followed by 200 mg weekly for 5 weeks, and (iii) 400 mg weekly for 6 weeks. Percent probabilities of achieving the nonclinical PK-PD targets associated with net fungal stasis and 1-log10 CFU reductions from baseline for Candida albicans and Candida glabrata were calculated for each rezafungin regimen. At the MIC90 for C. albicans and C. glabrata, a single 400 mg dose of rezafungin achieved probabilities of PK-PD target attainment of ≥90% through week 3 of therapy for all PK-PD targets evaluated. When evaluating the multiple-dose (i.e., weekly) regimens under these conditions, percent probabilities of PK-PD target attainment of 100% were achieved through week 6. Moreover, high (>90%) probabilities of PK-PD target attainment were achieved through week 6 following administration of the weekly regimens at or above the MIC100 values for C. albicans and C. glabrata based on contemporary in vitro surveillance data. These analyses support the use of single and once-weekly rezafungin regimens for the treatment of patients with candidemia and/or candidiasis due to C. albicans or C. glabrata.

Pharmacoeconomic analysis of antifungal therapy for primary treatment of invasive candidiasis caused by Candida albicans and non-albicans Candida species.

BACKGROUND: Cost-effectiveness studies of echinocandins for the treatment of invasive candidiasis, including candidemia, are rare in Asia. No study has determined whether echinocandins are cost-effective for both Candida albicans and non-albicans Candida species. There have been no economic evaluations that compare non-echinocandins with the three available echinocandins. This study was aimed to assess the cost-effectiveness of individual echinocandins, namely caspofungin, micafungin, and anidulafungin, versus non-echinocandins for C. albicans and non-albicans Candida species, respectively. METHODS: A decision tree model was constructed to assess the cost-effectiveness of echinocandins and non-echinocandins for invasive candidiasis. The probability of treatment success, mortality rate, and adverse drug events were extracted from published clinical trials. The cost variables (i.e., drug acquisition) were based on Taiwan's healthcare system from the perspective of a medical payer. One-way sensitivity analyses and probability sensitivity analyses were conducted. RESULTS: For treating invasive candidiasis (all species), as compared to fluconazole, micafungin and caspofungin are dominated (less effective, more expensive), whereas anidulafungin is cost-effective (more effective, more expensive), costing US$3666.09 for each life-year gained, which was below the implicit threshold of the incremental cost-effectiveness ratio in Taiwan. For C. albicans, echinocandins are cost-saving as compared to non-echinocandins. For non-albicans Candida species, echinocandins are cost-effective as compared to non-echinocandins, costing US$652 for each life-year gained. The results were robust over a wide range of sensitivity analyses and were most sensitive to the clinical efficacy of antifungal treatment. CONCLUSIONS: Echinocandins, especially anidulafungin, appear to be cost-effective for invasive candidiasis caused by C. albicans and non-albicans Candida species in Taiwan.

Assessment of silibinin as a potential antifungal agent and investigation of its mechanism of action.

Silibinin, which is derived from Silybum marianum (milk thistle), has used as a traditional remedy for liver or biliary disorders and known to have superior antioxidant activity. In addition, silibinin was recently reported to have antifungal effect related to fungal apoptosis against Candida albicans and the interest in the therapeutic effect is increasing. In this study, we found another mode of antifungal action of silibinin and its antibiofilm activity on C. albicans. To investigate influence on fungal plasma membrane, propidium iodide and bis-(1, 3-dibutylbarbituric acid) trimethineoxonol [DiBAC4 (3)] assay were primarily carried out. After 5-h incubation with silibinin (50, 100, 150, or 200 µg/mL), the propidium iodide fluorescent percentages increased by 11.90%, 28.50%, 34.10%, and 44.52%, respectively, and the DiBAC4 (3) fluorescent percentages increased by 13.18%, 34.64%, 46.99%, and 57.15%, respectively. As a result, we thought that silibinin concentrations of more than 100 µg/mL have a membrane-damaging effect. Subsequently, to estimate the degree of membrane damage, we used Fluorescein isothiocyanate-labelled dextrans (FDs) of various sizes and the results indicated that silibinin allowed penetration of molecules smaller than approximately FD20 (3.3 nm). In addition, silibinin inhibited the dimorphic transition of C. albicans and resulted in the inhibition of biofilm development at an early stage. In conclusion, we found membrane-damaging effect of silibinin and its antibiofilm effect in C. albicans. © 2017 IUBMB Life, 69(8):631-637, 2017.

Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays.

Candida albicans biofilms have a significant medical impact due to their rapid growth on implanted medical devices, their resistance to antifungal drugs, and their ability to seed disseminated infections. Biofilm assays performed in vitro allow for rapid, high-throughput screening of gene deletion libraries or antifungal compounds and typically serve as precursors to in vivo studies. Here, we compile and discuss the protocols for several recently published C. albicansin vitro biofilm assays. We also describe improved versions of these protocols as well as novel in vitro assays. Finally, we consider some of the advantages and disadvantages of these different types of assays.

A virtual infection model quantifies innate effector mechanisms and Candida albicans immune escape in human blood.

Candida albicans bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against C. albicans, fungal cells were added to human whole-blood samples. After inoculation, C. albicans started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost [Formula: see text] of C. albicans killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against C. albicans in human blood. A fraction of C. albicans cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of C. albicans cells being resistant against phagocytosis may account for the high proportion of dissemination in C. albicans bloodstream infection. Taken together, iterative experiment-model-experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.

Stage specific assessment of Candida albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants.

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.

Resistance to echinocandins comes at a cost: the impact of FKS1 hotspot mutations on Candida albicans fitness and virulence.

The emergence of Candida strains carrying FKS1 hotspot mutations associated with resistance to echinocandins is cause for concern. However, to assess the potential of such strains to spread within the community and cause lethal infection, the impact of FKS1 mutations on Candida fitness must be determined. We present evidence that C. albicans fks1 mutations carry significant fitness and virulence costs, which are associated with the production of a thickened, chitin-rich cell wall, impaired filamentation and induction of a dampened inflammatory response. If these phenotypic changes remain stable, they can serve as a basis for rational design of strategies to control the spread of echinocandin resistance.

Fitness and virulence costs of Candida albicans FKS1 hot spot mutations associated with echinocandin resistance.

The identification of FKS1 mutations in Candida albicans associated with echinocandin resistance has raised concerns over the spread of drug-resistant strains. We studied the impact of fks1 mutations on C. albicans virulence and fitness. Compared with wild-type strains for FKS1, echinocandin-resistant C. albicans strains with homozygous fks1 hot-spot mutations had reduced maximum catalytic capacity of their glucan synthase complexes and thicker cell walls attributable to increased cell wall chitin content. The fks1 mutants with the highest chitin contents had reduced growth rates and impaired filamentation capacities. Fks1 mutants were hypovirulent in fly and mouse models of candidiasis, and this phenotype correlated with the cell wall chitin content. In addition, we observed reduced fitness of echinocandin-resistant C. albicans in competitive mixed infection models. We conclude that fks1 mutations that confer echinocandin resistance come at fitness and virulence costs, which may limit their epidemiological and clinical impact.

Assessment of Candida albicans genes expressed during infections as a tool to understand pathogenesis.

Candida albicans is the most common fungal opportunistic pathogen of humans and causes mucocutaneous, bloodstream and deep organ infections. Screening for C. albicans genes that are preferentially expressed within infected hosts represents a strategy to identify novel virulence factors and define global expression patterns relevant to pathogenesis. Until recently, C. albicans has not been amenable to screening using existing technologies. This has begun to change with the development of new molecular genetic tools and the sequencing of the C. albicans genome. In this paper, we review studies using recently developed techniques to identify genes expressed by C. albicans during infections, as well as work from our laboratory using a human antibody-based strategy. Along with others, we have shown that selected in vivo expressed genes encode known and previously unrecognized candidal virulence factors. Future studies in this area will identify additional novel virulence factors, as well as advance our understanding of pathogenesis.

URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes.

The ability to generate isogenic sets of strains with mutations in a gene of interest but not in other genes by repeated use of the URA3 marker (Ura-blaster methodology) has advanced our understanding of the relationships between gene structure and function in Candida albicans. Common applications of Ura-blaster technology result in different genomic positions for the URA3 gene in strains complemented for the gene of interest compared with mutant strains. Studies using animal models of systemic candidiasis pointed to possible differences in URA3 gene expression, depending on its genomic location, which confounded interpretation of the role of the gene of interest in lethality. Positional effects on URA3 expression can be avoided by placement at a common locus in all strains used for comparison.

Utilidad del frotis faríngeo en pacientes con neutropenia febril / Assessment of pharinx frotis in patients with fever neutropenia

El frotis faríngeo (FF) está entre los múltiples exámenes que se toman a todo paciente que ingresa con el diagnóstico de neutropenia febril (NF) sin foco infeccioso aparente. Se evaluó su utilidad mediante una investigación de tipo retrospectivo. Se obtuvo el resultado de todos los FF tomados a pacientes con el diagnóstico de NF durante los años 1993 y 1994 de los registros de bacteriología del Laboratorio Central del Hospital Roberto del Río. Luego se revisó la ficha clínica de los pacientes a los que se tomó FF buscando si el resultado estaba archivado,si se comentó en la evolución médica o si en base a él se cambió el esquema antibiótico. Se obtuvo 200 cultivos de FF tomados los años 1993 y 1994. La mayoría de ellos (78 por ciento) presentó flora faríngea habitual exclusiva. Un 6,5 por ciento presentó algún comensal oportunista entre los gérmenes obtenidos y en un 15 por ciento se cultivó uno o más gérmenes potencialmente patógenos (ninguno con cuadro clínico compatible con faringoamigdalitis o mucositis). En un 5,5 por ciento se aisló gérmenes propios de pacientes inmunodeprimidos (Candia albicans). Los 200 cultivos correspondían a 119 pacientes. Se lograron recolectar 98 fichas (82,35 por ciento) lo que permitió analizar 181 cultivos (90,5 por ciento del total). Se encontró que el examen estaba archivado en un 14,36 por ciento. Nunca el resultado fue comentado en la evolución médica y nunca determinó cambios en la conducta antibiótica. En base a esto podemos concluir que el FF tomado a pacientes que ingresan con NF sin foco aparente tiene una baja utilidad. Tal vez su uso se deba limitar a aquellos casos con antecedentes de malestar faríngeo o con elementos al examen físico sugerentes de mucositis o faringoamigdalitis

[Distribution of epitheliocytes by the number of Candida albicans adhered to them and assessment of the intensity of the adhesion in an in vitro test]. / Raspredelenie épiteliotsitov po chislu adgezirovannykh na nikh kletok Candida albicans i otsenka intensivnosti adgezii v teste in vitro.

The study of the adhesion test in suspensions, carried out with 9 C. albicans strains and human buccal epithelial cells, has revealed that these cells are exponentially distributed by the number of C. albicans cells adhered to them. To characterize adhesion, the use of the index of the exponent describing the distribution of epithelial cells is proposed. With the use of this index, 5 C. albicans strains isolated from candidiasis patients have been found to possess, on the average, higher adhesiveness with respect to epithelial cells than 4 strains isolated from persons without symptoms of candidiasis.