In vitro biofilm production of Candida bloodstream isolates: any association with clinical characteristics?

Candida spp. are a leading cause of bloodstream infection (BSI) and are associated with high mortality rates. Biofilm production is a virulence factor of Candida spp., and has been linked with poor clinical outcome. The aim of our study was to assess biofilm production of Candida bloodstream isolates at our institute, and to determine whether in vitro biofilm production is associated with any clinical characteristics of infection. During the four-year study period, 93 cases of Candida BSI were analysed. The most frequently isolated species was C. albicans (66.7 %), followed by C. glabrata (9.7 %), C. parapsilosis (9.7 %), C. tropicalis (9.7 %) and C. krusei (4.3 %). Biofilm production was more prevalent among non-albicans Candida spp. (77.4 %) than C. albicans (30.6 %) (P = 0.02). Abdominal surgery was identified as a risk factor of BSI caused by biofilm producing non-albicans Candida isolates. No risk factors predisposing to bloodstream infection caused by a biofilm producing C. albicans isolate were identified. Biofilm production was not verified as a risk factor of mortality.

A critical review of analytical methods for subvisible and visible particles.

The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

Studies on the use of needle-free injection device on proteins.

In the following communication we report the evaluation of 18 proteins that were processed by a specific needle free injection device. The processed protein samples were analyzed by two HPLC techniques, reversed-phase liquid chromatography (RPLC) and size-exclusion chromatography (SEC). These techniques are two of the most widely used analytical techniques in the biopharmaceutical industry for the characterization, integrity assessment and stability study of peptide and protein products. The results indicate that needle free injection, using the specific device of this study, is not damaging to the studied proteins and does not generate aggregates. We found no evidence of the predicted possible effects of needle free injections, and concluded that needle free delivery is in general not different than any other delivery system and that its use should be evaluated on a case by case basis. It has to be noted that there are various needle free device designs and our work was performed using an Iject from Bioject. Our conclusions therefore should be limited to the Iject design we used in this study. In the reported experiments we used commercially available (economical) model proteins, which facilitate the use of the results for future comparison and reference. The work reported here can serve as a reference to illustrate the benign nature of our needle free injection device. It also highlights an interesting analogy between a set of phobias that were seen to have plagued the early stages of biochemistry and HPLC, on the one hand, and some attitudes that appear to hinder the widespread acceptance of needle free injection at present time, on the other. These phobias were identified and named by Professor Csaba Horváth, the father of HPLC, as barophobia, siderophobia and lithophobia. Today a wealth of evidence is available to indicate that those phobias are ungrounded and that the negative observations can be explained in most cases by adsorption and prevented by proper formulations and solvent conditions.