Phylogeny of dermatophytes with genomic character evaluation of clinically distinct Trichophyton rubrum and T. violaceum.

Trichophyton rubrum and T. violaceum are prevalent agents of human dermatophyte infections, the former being found on glabrous skin and nail, while the latter is confined to the scalp. The two species are phenotypically different but are highly similar phylogenetically. The taxonomy of dermatophytes is currently being reconsidered on the basis of molecular phylogeny. Molecular species definitions do not always coincide with existing concepts which are guided by ecological and clinical principles. In this article, we aim to bring phylogenetic and ecological data together in an attempt to develop new species concepts for anthropophilic dermatophytes. Focus is on the T. rubrum complex with analysis of rDNA ITS supplemented with LSU, TUB2, TEF3 and ribosomal protein L10 gene sequences. In order to explore genomic differences between T. rubrum and T. violaceum, one representative for both species was whole genome sequenced. Draft sequences were compared with currently available dermatophyte genomes. Potential virulence factors of adhesins and secreted proteases were predicted and compared phylogenetically. General phylogeny showed clear gaps between geophilic species of Arthroderma, but multilocus distances between species were often very small in the derived anthropophilic and zoophilic genus Trichophyton. Significant genome conservation between T. rubrum and T. violaceum was observed, with a high similarity at the nucleic acid level of 99.38 % identity. Trichophyton violaceum contains more paralogs than T. rubrum. About 30 adhesion genes were predicted among dermatophytes. Seventeen adhesins were common between T. rubrum and T. violaceum, while four were specific for the former and eight for the latter. Phylogenetic analysis of secreted proteases reveals considerable expansion and conservation among the analyzed species. Multilocus phylogeny and genome comparison of T. rubrum and T. violaceum underlined their close affinity. The possibility that they represent a single species exhibiting different phenotypes due to different localizations on the human body is discussed.

Simple and rapid characterization of novel large germline deletions in SDHB, SDHC and SDHD-related paraganglioma.

Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with hereditary paraganglioma and pheochromocytoma. Although most mutations in SDHB, SDHC and SDHD are intraexonic variants, large germline deletions may represent up to 10% of all variants but are rarely characterized at the DNA sequence level. Additional phenotypic effects resulting from deletions that affect neighboring genes are also not understood. We performed multiplex ligation-dependent probe amplification, followed by a simple long-range PCR 'chromosome walking' protocol to characterize breakpoints in 20 SDHx-linked paraganglioma-pheochromocytoma patients. Breakpoints were confirmed by conventional PCR and Sanger sequencing. Heterozygous germline deletions of up to 104 kb in size were identified in SDHB, SDHC, SDHD and flanking genes in 20 paraganglioma-pheochromocytoma patients. The exact breakpoint could be determined in 16 paraganglioma-pheochromocytoma patients of which 15 were novel deletions. In six patients proximal genes were also deleted, including PADI2, MFAP2, ATP13A2 (PARK9), CFAP126, TIMM8B and C11orf57. These genes were either partially or completely deleted, but did not modify the phenotype. This study increases the number of known SDHx deletions by over 50% and demonstrates that a significant proportion of large gene deletions can be resolved at the nucleotide level using a simple and rapid method.

Development of a species-specific PCR-restriction fragment length polymorphism analysis procedure for identification of Madurella mycetomatis.

Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomatis and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of M. mycetomatis, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with CfoI, HaeIII, MspI, Sau3AI, RsaI, and SpeI restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.

Ciprofibrate versus gemfibrozil in the treatment of primary hyperlipidaemia.

The efficacy and short-term safety of ciprofibrate and gemfibrozil were compared in a 12-week, double-blind, randomised study. One-hundred-and-ten primary, type II hyperlipidaemic patients were randomised to receive either ciprofibrate, 100 mg/day once daily, or gemfibrozil, 1200 mg/day twice daily. Treatment efficacy was measured by complete lipid and lipoprotein profiles and by plasma fibrinogen levels. Tolerability was assessed by drug compliance and safety was evaluated by laboratory safety parameters, physical examination and evaluation of adverse events. Mean reductions of plasma TC and low density lipoprotein cholesterol levels were similar in the two treatment groups. In contrast, the mean relative reduction of plasma total triglyceride and very low density lipoprotein triglyceride levels was significantly higher in patients receiving gemfibrozil as compared with ciprofibrate (P < 0.05). The absolute reduction of the last two parameters was higher in the ciprofibrate group compared with the gemfibrozil group; furthermore, the mean concentrations of these parameters were within normal limits at the end of the study. The clinical relevance of the statistically significant difference mentioned should, therefore, be questioned. Ciprofibrate therapy significantly reduced (-8.33%) and gemfibrozil therapy significantly increased (+6.97%) plasma fibrinogen levels (P < 0.001 compared with baseline in each case). Adverse events were rare, mild and equally distributed between the two treatment groups. Laboratory safety parameters did not show any significant changes. Ciprofibrate and gemfibrozil have comparable short-term efficacy and safety profiles. Furthermore, ciprofibrate reduces fibrinogen levels and benefits from a once daily regimen.

The role of plasma D-dimers concentration in the exclusion of pulmonary embolism.

OBJECTIVE: To determine the role of four ELISA D-dimer assays in the exclusion of pulmonary embolism. DESIGN: Blinded comparison using pulmonary angiography and/or lung scintigraphy as a reference method. SETTING: A secondary and tertiary referral centre. PATIENTS AND METHODS: Consecutive patients with suspected pulmonary embolism underwent lung scintigraphy, followed by angiography if a non-diagnostic result was obtained. Comorbid conditions resulting in elevated plasma D-dimer levels were defined a priori. Cut-off levels for 100% sensitivity was determined. A decision-analytic model was used to determine effectiveness and costs in the management pulmonary embolism. MAIN OUTCOME MEASURES: The exclusion efficacy of the various assays at a sensitivity of 100% and cost-effectiveness. RESULTS: A total of 179 patients were included (78 inpatients and 101 outpatients; 74 patients had comorbid conditions). Pulmonary embolism could be adequately excluded in between 8% and 18% of all patients, an in between 3% and 7% and 11% and 27% of inpatients and outpatients, respectively, depending on the assay used. D-dimer assays could exclude pulmonary embolism in <5% of patients with comorbid conditions, whereas this increased to 14-32% in outpatients without comorbid conditions. A cost-effectiveness analysis showed a cost reduction of 10% at a specificity of 30%, largely due to a 28% decrease in angiography requirements. Furthermore, for every 2% decrease to sensitivity, one per 1000 evaluated patients would die as a result of inadequately treated pulmonary embolism. CONCLUSION: D-dimer ELISA assays may have a role in the exclusion of pulmonary embolism in symptomatic outpatients, where the application may reduce angiography by 30% and costs by 10%.

Inhibition of endotoxin-induced activation of the coagulation and fibrinolytic pathways using a recombinant endotoxin-binding protein (rBPI23).

A recombinant endotoxin-neutralizing protein, rBPI23, was shown to partially prevent endotoxin-induced activation of the fibrinolytic and coagulation systems in experimental endotoxemia in humans. In a placebo-controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg of body weight) and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). rBPI23 treatment significantly lowered the endotoxin-induced fibrinolytic response, ie, reduced the release of tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor antigen, and complex formation of plasmin alpha 2-antiplasmin (P = .0078 for each). Plasminogen activator inhibitor activity was also reduced, but not significantly according to the Hochberg method (P = .0304). The endotoxin-induced activation of the procoagulant state as reflected by increase in F1 + 2 fragments and TAT complexes was blunted by rBPI23 infusion (P = .0391 [not significant according to the Hochberg method] and .0078, respectively). These results indicate that rBPI23 is capable of reducing both the activation of the fibrinolytic and the coagulation systems after low-dose endotoxin infusion in humans.

A comparative analysis of D-dimer assays in patients with clinically suspected pulmonary embolism.

To avoid angiography in patients with clinically suspected pulmonary embolism and non-diagnostic lung scan results, the use of D-dimer has been advocated. We assessed plasma samples of 151 consecutive patients with clinically suspected pulmonary embolism. Lung scan results were: normal (43), high probability (48) and non-diagnostic (60; angiography performed in 43; 12 pulmonary emboli). Reproducibility, cut-off values, specificity, and percentage of patients in whom angiography could be avoided (with sensitivity 100%) were determined for two latex and four ELISA assays. The latex methods (cut-off 500 micrograms/l) agreed with corresponding ELISA tests in 83% (15% normal latex, abnormal ELISA) and 81% (7% normal latex, abnormal ELISA). ELISA methods showed considerable within- (2-17%) and between-assay variation (12-26%). Cut-off values were 25 micrograms/l (Behring), 50 micrograms/l (Agen), 300 micrograms/l (Stago) and 550 micrograms/l (Organon). Specificity was 14-38%; in 4-15% of patients angiography could be avoided. We conclude that latex D-dimer assays appear not useful, whereas ELISA methods may be of limited value in the exclusion of pulmonary embolism.

The effect of (2-chloroethyl)phosphonic acid on the sink strength of developing peach (Prunus persica L.) fruit.

The sink strength of one of a pair of competing peach fruits was increased when the fruit was treated with (2-chloroethyl)phosphonic acid (Ethephon) and gibberellic acid. Ethephon increased the capacity of the treated fruit to attract (14)C-labelled assimilates at most stages of fruit development and was most effective when the level of endogenous ethylene produced by the fruit was lowest. The results are discussed in relation to the hypothesis that ethylene participates in the control of sink strength of the fruit and of other competing organs of the tree.