Determination of ATP-activity as a useful tool for monitoring microbial load in aqueous humidifier samples.

Air humidifier water tanks are potential sources of microbial contaminants. Aerosolization of these contaminants is associated with the development of airway and lung diseases; therefore, implementation of preventive strategies including monitoring of the microbial contamination is recommended. So far, culture-based methods that include measuring colony forming units (CFU) are widely used to monitor microbial load. However, these methods are time consuming and have considerable drawbacks. As a result, alternative methods are needed which provide not only clear and accurate results concerning microbial load in water samples, but are also rapid and easy to use in the field. This paper reports on a rapid test for ATP quantification as an alternative method for microbial monitoring, including its implementation, validation and application in the field. For this purpose, 186 water samples were characterized with different methods, which included ATP analysis, culture-based methods, endotoxin activity (common and rapid test), pyrogenic activity and number of particles. Half of the samples was measured directly in the field and the other half one day later in the laboratory. The results of both tests are highly correlated. Furthermore, to check how representative the result from one sample of a water source is, a second sample of the same water tank were collected and measured. Bioluminescence results of the undiluted samples covered a range between 20 and 25,000 relative light units (RLU) and correlated with the results obtained using the other methods. The highest correlation was found between bioluminescence and endotoxin activity (rs=0.79) as well as pyrogenic activity (rs=0.75). Overall, the results of this study indicate that ATP measurement using bioluminescence is a suitable tool to obtain rapid, reproducible and sensitive information on the microbial load of water samples, and is suitable to use in the field. However, to use ATP measurement as an indicator of water quality, criteria of assessment has to be discussed.

Assessment of airborne exposure to endotoxin and pyrogenic active dust using electrostatic dustfall collectors (EDCs).

Passive airborne dust sampling using electrostatic dustfall collectors (EDCs) is one possibility especially for long sampling periods. In this study, EDCs were deposited in living rooms of private households and in social rooms of composting plants. The aim of the study was to determine whether endotoxin and pyrogenic activity are measurable using EDCs. In all extracts, endotoxin (via Limulus amebocyte lysate [LAL] assay) and pyrogenic activity (interleukin [IL]-1ß release via whole blood assay) were detectable. In addition, the monocyte chemotactic protein (MCP-1; CCL-2) as a secondary proinflammatory marker was measured with whole blood assay. Endotoxin activity and proinflammatory/pyrogenic activity of EDC extracts from social rooms in composting plants were higher compared to extracts obtained from EDCs in private household rooms. A significant correlation between LAL assay and whole blood assay was detectable. In conclusion, EDC sampling is an applicable method to evaluate settled dust from airborne bioaerosols displaying a longer period of exposure. The extraction of EDC without Tween enables one to measure endotoxin as well as proinflammatory/pyrogenic activity using the same sample for parallel detection and more reliable characterization of the airborne bioaerosol contamination.

Impact of different welding techniques on biological effect markers in exhaled breath condensate of 58 mild steel welders.

Total mass and composition of welding fumes are predominantly dependent on the welding technique and welding wire applied. The objective of this study was to investigate the impact of welding techniques on biological effect markers in exhaled breath condensate (EBC) of 58 healthy welders. The welding techniques applied were gas metal arc welding with solid wire (GMAW) (n=29) or flux cored wire (FCAW) (n=29). Welding fume particles were collected with personal samplers in the breathing zone inside the helmets. Levels of leukotriene B(4) (LTB(4)), prostaglandin E(2) (PGE(2)), and 8-isoprostane (8-iso-PGF(2α)) were measured with immunoassay kits and the EBC pH was measured after deaeration. Significantly higher 8-iso-PGF(2α) concentrations and a less acid pH were detected in EBC of welders using the FCAW than in EBC of welders using the GMAW technique. The lowest LTB(4) concentrations were measured in nonsmoking welders applying a solid wire. No significant influences were found in EBC concentrations of PGE(2) based upon smoking status or type of welding technique. This study suggests an enhanced irritative effect in the lower airways of mild steel welders due to the application of FCAW compared to GMAW, most likely associated with a higher emission of welding fumes.

Standardization of whole blood assay for determination of pyrogenic activity in organic dust samples.

To characterize bioaerosol exposure at workplaces standardized methods are necessary. Activity of endotoxin, one component of organic dust, can be quantified with the Limulus-Amoebocyte Lysat test (LAL test). Further information with respect to pyrogenic activity of the organic dust can be achieved by measuring cytokine release of human blood after stimulation with the dust or its extract (whole blood assay). The aim of our study was the standardization of the whole blood assay (WBA) while using cryo-preserved human blood (Qualis Laboratorium) and to compare the outcome of the different cytokines determined by incubation of the blood cells with extracts from dust samples collected at various workplaces. Cytokine release (IL-1 beta, IL-6, IL-8, TNF-alpha, MCP-1) was measured by ELISA after stimulation of fresh blood from ten donors as well as cryo-preserved human blood. In both cases blood was stimulated with E. coli endotoxin as well as with 30 dust filter extracts from various workplaces. All dust filter extracts were investigated in the WBA using cryo-preserved blood as well as with LAL test. E. coli endotoxin stimulated the release of IL-1 beta, IL-6, IL-8, TNF-alpha and MCP-1 in a dose-dependent manner in fresh as well as cryo-preserved human whole blood. 200 pg/ml E. coli endotoxin induced maximal cytokine release in cryo-preserved blood (mean value for IL-1 beta 2509+/-418 pg/ml; n=13 experiments) whereas fresh blood of single donors reached a maximum release when stimulated with 50 ng/ml endotoxin (mean value of ten donors 1125+/-553 pg/ml IL-1beta). Using cryo-preserved blood the coefficient of variation (CV) regarding the interassay variability was below 21% for all cytokines measured. Regarding 26 dust sample extracts correlation coefficient r2 for LAL test and WBA was between 0.90 and 0.93 (Pearson) for IL-1 beta, IL-6, IL-8 and TNF-alpha whereas correlation for MCP-1 was lower (r(2)=0.59). Two dust sample extracts which showed similar reactivity patterns in LAL test as well as in WBA with respect to IL-1 beta, IL-6, IL-8 and TNF-alpha could be differentiated by measuring MCP-1. In conclusion, cryo-preserved blood pools are suitable to standardize WBA. Combination of different outcome variables like IL-1 beta and MCP-1 improve the characterization from the inflammatory potency of workplace related dust samples.