No abstract

We describe an analytical protocol to study biofilms that develop inside silicone tubing of dialysis machines. This protocol has been set up with the help of a dynamic testing device reproducing dialysis conditions. The methodology includes direct microscopic observation, biofilm removal with an original mechanical biofilm scraper, quantitative analysis with culturable and total bacteria counting, and endotoxin level measurement using the LAL chromogenic kinetic assay. The analytical protocol has been assessed on 13 different clinical tubing samples. Most samples were contaminated by adherent cells and the thickest biofilms were found at the connection between the dialysis water distribution loop and the dialysis machine. The less contaminated samples had been removed from dialysis machines that were decontaminated with citric acid and autoclaving, showing the importance of the decontamination procedure for the prevention of biofilm development. This article shows that easy, rapid, reproducible, and economical methods are applicable for a routine analysis of biofilms that develop on dialysis systems and should be included in the regular control of the microbiological quality of dialysis liquids.

Biofilms have been found on the inner surface of silicone tubing inside dialysis machines. Endotoxin releasing from those biofilms increases the bioincompatibility of dialysis liquids and leads to long-term inflammatory complications among dialysis patients. Endotoxin measurement is recommended for the control of dialysis liquids. This article describes the use of a new method, the Whole Blood Assay (WBA), for endotoxin quantification in dialysis biofilms. Biofilms were suspended in sterile water by scraping the tubing samples. Diluted blood samples from healthy donors were stimulated overnight with the contaminated suspension. Stimulated mononuclear cells released IL-1beta in response to endotoxins. IL-1beta level was then measured using an ultrasensitive ELISA method. We demonstrated a semilogarithmic model in which the optical densities measured after the ELISA assay increases linearly with the levels of endotoxin. This model allowed the determination of the amount of endotoxins in biofilm samples with a detection limit of 0.032 EU/mL. Most of the time, the amounts of endotoxin measured by the WBA were higher than those measured by the Limulus Amoebocyte Lysate (LAL) assay. This study suggested the presence of "endotoxin-like" compounds different from the lipopolysaccharides that are not detected by the LAL assay. We concluded that the LAL is necessary but insufficient to have a representative quantification of endotoxins that could be hazardous to patient health.