A Novel Diagnostic Target in the Hepatitis C Virus Genome

BackgroundDetection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (59-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.Methods and FindingsIn this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.ConclusionThis study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Actinobacillus actinomycetemcomitans lipopolysaccharide-mediated experimental bone loss model for aggressive periodontitis

Background: Bacterial constituents, such as Gram-negative derived lipopolysaccharide (LPS), can initiate inflammatory bone loss through induction of host-derived inflammatory cytokines. The aim of this study was to establish a model of aggressive inflammatory alveolar bone loss in rats using LPS derived from the periodontal pathogen Actinobacillus actinomycetemcomitans.Methods: Eighteen female Sprague-Dawley rats were divided into LPS test (N = 12) and saline control (N = 6) groups. All artimals received injections to the palatal molar gingiva three times per week for 8 weeks. At 8 weeks, linear and volumetric alveolar bone loss was measured by micro-computed tomography (mu CT). The prevalence of inflammatory infiltrate, proinflammatory cytokines, and osteoclasts was assessed from hematoxylin and eosin, immunohistochemical, or tartrate-resistant acid phosphatase (TRAP)-stained sections. Statistical analysis was performed.Results: A. actinomycetemcomitans LPS induced severe bone loss over 8 weeks, whereas control groups were unchanged. Linear and volumetric analysis of maxillae by mu CT indicated significant loss of bone with LPS, administration. Histologic examination revealed increased inflammatory infiltrate, significantly increased immunostaining for interleukin IL-6 and -1 beta and tumor necrosis factor-alpha, and more TRAP-positive osteoclasts in the LPS group compared to controls.Conclusion: Oral injections of LPS derived from the periodontal pathogen A. actinomycetemcomitans can induce severe alveolar bone loss and proinflammatory cytokine production in rats by 8 weeks.

Seed dispersal by fishes in tropical and temperate fresh waters: The growing evidence

Fruit-eating by fishes represents an ancient (perhaps Paleozoic) interaction increasingly regarded as important for seed dispersal (ichthyochory) in tropical and temperate ecosystems. Most of the more than 275 known frugivorous species belong to the mainly Neotropical Characiformes (pacus, piranhas) and Siluriformes (catfishes), but cypriniforms (carps, minnows) are more important in the Holarctic and Indomalayan regions. Frugivores are among the most abundant fishes in Neotropical floodplains where they eat the fruits of a wide variety of trees and shrubs. By consuming fruits, fishes gain access to rich sources of carbohydrates, lipids and proteins and act as either seed predators or seed dispersers. With their often high mobility, large size, and great longevity, fruit-eating fishes can play important roles as seed dispersers and exert strong influences on local plant-recruitment dynamics and regional biodiversity. Recent feeding experiments focused on seed traits after gut passage support the idea that fishes are major seed dispersers in floodplain and riparian forests. Overfishing, damming, deforestation and logging potentially diminish ichthyochory and require immediate attention to ameliorate their effects. Much exciting work remains in terms of fish and plant adaptations to ichthyochory, dispersal regimes involving fishes in different ecosystems, and increased use of nondestructive methods such as stomach lavage, stable isotopes, genetic analyses and radio transmitters to determine fish diets and movements. (C) 2011 Elsevier Masson SAS. All rights reserved.