New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction

Kinetics of Expansion of Human Limbal Epithelial Progenitor Cells in Primary Culture of Explants Without Feeders

International audienceThe aims of this study were to determine whether human limbal explant cultures without feeder cells result in expansion of epithelial progenitors and to estimate the optimal expansion time for progenitor cells. Limbal explants from ten human corneas were cultured for 7, 9, 11, 14, 18, and 21 days. Limbal explants from two corneas were enzymatically dissociated or directly cultured for 14 days. Progenitor cells were characterized by their ability to form colonies, by immunocytochemistry, and by quantitative real-time polymerase chain reaction. Colonies were identified after 9, 11, 14, and 18 days of culture, but not after 21 days. The number of colonies per explant was significantly higher after 14 days than after 9 and 21 days. The mean percentage of seeded cells giving rise to clones was 4.03% after 14 days of culture and 0.36% for non-cultured dissociated limbal epithelial cells. The number of cells giving rise to clones per cornea significantly increased from an average of 2275 for non-cultured cells to 24266 for cells cultured for 14 days. Immunocytochemical analysis detected positive staining for cytokeratin (CK) 3, CK5/6/8/10/13/18, CK19, vimentin, p63, and p63α, in both cultures and clones. CK3 expression increased significantly with culture time. Transcript expression was observed for CK3, CK19, vimentin, and Delta N p63α at each culture time point, both in cultures and clones. The optimal culture time for limbal explants in cholera toxin-free Green medium without feeder cells was 14 days leading to the expansion of progenitors

Experimental design.

<p><b>A</b>. First series of experiments: 60 human superficial limbal explants were sutured on a tissue-culture treated round coverslip epithelium side up and cultured for 7 to 21 days using 6-well plates. <b>B</b>. Second series of experiments: 6 superficial limbal explants from two human donor corneas (3 per cornea) were sutured and cultured for 14 days and the remaining 6 explants were digested with collagenase A.</p

Number of cells after human limbal explant culture.

<p>Ten limbal rims retrieved from 10 human corneas were divided in 6 explants each and cultured for 7, 9, 11, 14, 18, or 21 days. Only trypan blue negative cells grown out of the explants were counted.</p

Clonal growth on a 3T3 feeder cell layer and colony-forming efficiency.

<p><b>A</b> Macroscopic appearance of colonies obtained from cells grown from explants cultured for 9, 11, 14, 18, and 21 days and stained with crystal violet. <b>B</b> Photograph of microscopic appearance of limbal stem cell (LSC) colonies. At the end of primary culture, cells were dissociated and cultured with mitomycin-arrested 3T3 murine feeder cells for 12 days (2 cultures for each primary culture). Colonies were assessed at the end of culture. <b>C</b> Significantly higher CFE (%) was obtained after 9, 11, and 14 days of primary culture compared with 21 days (p=0.00002; ANOVA). Shown are mean <u>+</u> standard errors of the mean and post-hoc tests. <b>D</b> The primary culture time significantly influenced the CFE (N) (number of cells obtained at the end of primary culture * number of clones / number of seeded cells). Significantly higher CFE (N) was obtained after 14 days of primary culture compared with 9 and 21 days. (p=0.004; ANOVA). Shown are mean <u>+</u> standard errors of the mean and post-hoc tests. </p

Comparison of dissociated limbal epithelial cells with cultured limbal epithelial cells for 14 days.

<p><b>A</b> Colony forming efficiency expressed as the percentage of seeded cells giving rise to clone (CFE %). Significantly higher CFE (%) (p=0.000002) was obtained after 14 days of primary culture of limbal explants compared with non-cultured dissociated limbal epithelial cells. <b>B, C</b> Immunohistochemical analysis of enzymatically dissociated cells and cells cultured for 14 days from human limbal explants. <b>B</b> Primary culture: expression of differentiation markers and progenitors markers was significantly higher in cells cultured for 14 days than in dissociated cells from the explants (p < 0.02) except for CK3 (p = 0.44). C Clones: immunostaining of differentiation and progenitor markers was significantly higher in colonies obtained from cells cultured for 14 days than in colonies obtained from cells dissociated from explants (p < 0.02). Shown are mean <u>+</u> standard errors of the mean.</p

Percentage of CK3-expressing cells after primary culture of human limbal explants for 9 to 21 days.

<p>CK3 expression increased with culture time from day 9 to day 21 (p= 0.009).</p

New tool for the simultaneous detection of ten genotypes of Acanthamoeba

International audienceNew tool for the simultaneous detection of ten different genotypes of Acanthamoeba available from the American Type Culture Collectio

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes

Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration

International audienceSeveral diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of Delta Np63 alpha and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%). (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved