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

How to use the world's scarce selenium resources efficiently to increase the selenium concentration in food

The world's rare selenium resources need to be managed carefully. Selenium is extracted as a by-product of copper mining and there are no deposits that can be mined for selenium alone. Selenium has unique properties as a semi-conductor, making it of special value to industry, but it is also an essential nutrient for humans and animals and may promote plant growth and quality. Selenium deficiency is regarded as a major health problem for 0.5 to 1 billion people worldwide, while an even larger number may consume less selenium than required for optimal protection against cancer, cardiovascular diseases and severe infectious diseases including HIV disease. Efficient recycling of selenium is difficult. Selenium is added in some commercial fertilizers, but only a small proportion is taken up by plants and much of the remainder is lost for future utilization. Large biofortification programmes with selenium added to commercial fertilizers may therefore be a fortification method that is too wasteful to be applied to large areas of our planet. Direct addition of selenium compounds to food (process fortification) can be undertaken by the food industry. If selenomethionine is added directly to food, however, oxidation due to heat processing needs to be avoided. New ways to biofortify food products are needed, and it is generally observed that there is less wastage if selenium is added late in the production chain rather than early. On these bases we have proposed adding selenium-enriched, sprouted cereal grain during food processing as an efficient way to introduce this nutrient into deficient diets. Selenium is a non-renewable resource. There is now an enormous wastage of selenium associated with large-scale mining and industrial processing. We recommend that this must be changed and that much of the selenium that is extracted should be stockpiled for use as a nutrient by future generations

Toxicity of chlorhexidine on odontoblast-like cells

Chlorhexidine gluconate (CHX) is recommended for a number of clinical procedures and it has been pointed out as a potential cavity cleanser to be applied before adhesive restoration of dental cavities. OBJECTIVE: As CHX may diffuse through the dentinal tubules to reach a monolayer of odontoblasts that underlies the dentin substrate, this study evaluated the cytotoxic effects of different concentrations of CHX on cultured odontoblast-like cells (MDPC-23). MATERIAL AND METHODS: Cells were cultured and exposed to CHX solutions at concentrations of 0.06%, 0.12%, 0.2%, 1% and 2%. Pure culture medium (&#945;-MEM) and 3% hydrogen peroxide were used as negative and positive control, respectively. After exposing the cultured cells to the controls and CHX solutions for 60 s, 2 h or 60 s with a 24-h recovery period, cell metabolism (MTT assay) and total protein concentration were evaluated. Cell morphology was assessed under scanning electron microscopy. CHX had a dose-dependent toxic effect on the MDPC-23 cells. RESULTS: Statistically significant difference was observed when the cells were exposed to CHX in all periods (p<0.05). Significant difference was also determined for all CHX concentrations (p<0.05). The 60-s exposure time was the least cytotoxic (p<0.05), while exposure to CHX for 60 s with a 24-h recovery period was the most toxic to the cells (p<0.05). CONCLUSION: Regardless of the exposure time, all CHX concentrations had a high direct cytotoxic effect to cultured MDPC-23 cells

The evolutionary history of the CD209 (DC-SIGN) family in humans and non-human primates

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K(A)/K(S) values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigen

Detection of novel papillomavirus in pigmented plaques of four pugs

Pugs are predisposed to the development of deeply pigmented, slightly elevated hyperkeratotic noncancerous plaques. Polymerase chain reaction amplification of a papillomavirus (PV)-like DNA fragment from such lesions suggested that PV may be responsible for them, although the predicted virus has not yet been identified. The goal of the present study was to make use of pigmented plaques from four pugs to identify and sequence the predicted virus. Taking advantage of the circular nature of PV DNA, the entire viral genome was amplified by rolling circle amplification and restriction enzyme analysis disclosed the same pattern in all four cases. Sequencing of one of the amplificates revealed a novel canine PV, termed CPV4, related to the recently described CPV3 but clearly distinct from canine oral PV and CPV2. Thus, a novel canine PV and a method for its future diagnosis are described