Dot Immunobinding Assay for the Rapid Serodetection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

Scedosporium; Cystic fibrosis; Serological detectionScedosporium; Fibrosi quística; Detecció serològicaScedosporium; Fibrosis quística; Detección serológicaThe detection of Scedosporium/Lomentospora is still based on non-standardized low-sensitivity culture procedures. This fact is particularly worrying in patients with cystic fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because a poor and delayed diagnosis can worsen the prognosis of the disease. To contribute to the discovery of new diagnostic strategies, a rapid serological dot immunobinding assay (DIA) that allows the detection of serum IgG against Scedosporium/Lomentospora in less than 15 min was developed. A crude protein extract from the conidia and hyphae of Scedosporium boydii was employed as a fungal antigen. The DIA was evaluated using 303 CF serum samples (162 patients) grouped according to the detection of Scedosporium/Lomentospora in the respiratory sample by culture, obtaining a sensitivity and specificity of 90.48% and 79.30%, respectively; positive and negative predictive values of 54.81% and 96.77%, and an efficiency of 81.72%. The clinical factors associated with the results were also studied using a univariate and a multivariate analysis, which showed that Scedosporium/Lomentospora positive sputum, elevated anti-Aspergillus serum IgG and chronic Pseudomonas aeruginosa infection were significantly associated with a positive result in DIA, while Staphylococcus aureus positive sputum showed a negative association. In conclusion, the test developed can offer a complementary, rapid, simple and sensitive method to contribute to the diagnosis of Scedosporium/Lomentospora in patients with CF.This research was funded by the Basque Government, grant numbers IT1362-19 and IT1657-22. L.M-S and M.A have received a predoctoral grant from the Basque Government and L.A-F from the University of the Basque Country (UPV/EHU)

Immunoproteomics-Based Analysis of the Immunocompetent Serological Response to <i>Lomentospora prolificans</i>

The filamentous fungus <i>Lomentospora prolificans</i> is an emerging pathogen causing severe infections mainly among the immunocompromised population. These diseases course with high mortality rates due to great virulence of the fungus, its inherent resistance to available antifungals, and absence of specific diagnostic tools. Despite being widespread in humanized environments, <i>L. prolificans</i> rarely causes infections in immunocompetent individuals likely due to their developed protective immune response. In this study, conidial and hyphal immunomes against healthy human serum IgG were analyzed, identifying immunodominant antigens and establishing their prevalence among the immunocompetent population. Thirteen protein spots from each morph were detected as reactive against at least 70% of serum samples, and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Hence, the most seroprevalent antigens were WD40 repeat 2 protein, malate dehydrogenase, and DHN1, in conidia, and heat shock protein (Hsp) 70, Hsp90, ATP synthase β subunit, and glyceraldehyde-3-phosphate dehydrogenase, in hyphae. More interestingly, the presence of some of these seroprevalent antigens was determined on the cell surface, as Hsp70, enolase, or Hsp90. Thus, we have identified a diverse set of antigenic proteins, both in the entire proteome and cell surface subproteome, which may be used as targets to develop innovative therapeutic or diagnostic tools

Morphological changes on <i>Lomentospora prolificans</i> cells caused by Voriconazole (VRC) exposure.

<p>Germination assays (A) were performed to analyze the effect of VRC on fungal cells. After 9 h of incubation cells were stained with calcofluor white and microscopically analysed (B) to determine their length (C), width (D), occupied area (E), and emitted fluorescence (F). Scale bar = 5 μm. Results are shown as mean ± SEM, n = 4. **p<0.01, ***p<0.0001 compared to non-treated cells. a.u., arbitrary units.</p

Effect of cell membrane- and wall-disturbing agents on <i>Lomentospora prolificans</i> in the presence of voriconazole.

<p>Decimal dilutions of conidial suspensions were spotted onto potato dextrose agar plates containing SDS (100 μg/ml), calcofluor white (500 μg/ml; CFW) or congo red (750 μg/ml; CR), and combined with 0, 2 or 4 μg/ml of voriconazole (VRC).</p

Ultrastructural analysis of voriconazole-induced changes on <i>Lomentospora prolificans</i> cells.

<p>Transmission electron microscopy images (A) and measurements of cell wall thickness (B) of non-treated and 2 μg/ml voriconazole-treated fungal cells. Black lines highlight the thickness of the outer fibrillar layer. Results are shown as mean ± SEM, n ≥ 20 cells. ***p<0.0001 compared to non-treated cells.</p

Biochemical characterization of the carbohydrate composition of <i>Lomentospora prolificans</i> cell wall.

<p>Carbohydrate compositional analysis of whole cell wall (A) and cell wall surface (B) upon exposure to 2 μg/ml voriconazole. Results are shown as mean ± SEM, n = 3. *p<0.05 compared to non-treated cells. Percentage of monosaccharide content in the whole cell wall (C) and surface (D).</p

Identification of the most differentially expressed proteins on <i>Lomentospora prolificans</i> cells exposed to voriconazole.

<p>Identification of the most differentially expressed proteins on <i>Lomentospora prolificans</i> cells exposed to voriconazole.</p

Effect of voriconazole on proteomic profiles of the <i>Lomentospora prolificans</i> cell surface subproteome.

<p>Fungal cells were grown in absence (A) or presence (B) of 2 μg/ml voriconazole, and their surfaceomes resolved by two-dimensional electrophoresis. Arrows point to the most differentially expressed protein spots that were identified by LC-MS/MS (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174885#pone.0174885.t001" target="_blank">Table 1</a>).</p

Microbiota and fungal-bacterial interactions in the cystic fibrosis lung

International audienceAbstract The most common genetic hereditary disease affecting Caucasians is cystic fibrosis (CF), which is caused by autosomal recessive mutations in the CFTR gene. The most serious consequence is the production of a thick and sticky mucus in the respiratory tract, which entraps airborne microorganisms and facilitates colonization, inflammation and infection. Therefore, the present article compiles the information about the microbiota and, particularly, the inter-kingdom fungal-bacterial interactions in the CF lung, the molecules involved and the potential effects that these interactions may have on the course of the disease. Among the bacterial compounds, quorum sensing-regulated molecules such as homoserine lactones, phenazines, rhamnolipids, quinolones and siderophores (pyoverdine and pyochelin) stand out, but volatile organic compounds, maltophilin and CF-related bacteriophages are also explained. These molecules exhibit diverse antifungal mechanisms, including iron starvation and induction of reactive oxygen and nitrogen species production. The fungal compounds are less studied, but they include cell wall components, siderophores, patulin and farnesol. Despite the apparent competition between microorganisms, the persistence of significant rates of bacterial-fungal co-colonization in CF suggests that numerous variables influence it. In conclusion, it is crucial to increase scientific and economic efforts to intensify studies on the bacterial-fungal inter-kingdom interactions in the CF lung

Study of the carbohydrate/protein composition in different strains and morphologies.

<p>(A) Carbohydrate/protein ratio in the crude extracts of the different strains of <i>Candida albicans</i>: Blastoconidia (□) and germ tubes (▪). The results shown correspond to the mean ± SD of three independent experiments. Statistically significant differences between different morphologies are indicated by two asterisks (**) (p<0.05). (B). Regression line for the B16 melanoma (B16M) cell adhesion to hepatic sinusoidal endothelial (HSE) cells induced by the strains and morphologies of <i>C. albicans</i> that lead a significant effect <i>versus</i> their carbohydrate/protein ratio.</p