A new bioluminescence method for the measurement of total adenosine triphosphate (ATP) was used to control the quality of clean rooms for the preparation of individual parenteral therapies, which is becoming increasingly used method for assessing the suitability of surfaces, including the hospital environment on intensive care units. Due to the fact that in the pharmaceutical production due to the undefined criteria of relevance and correlation with the existing methods of microbiological compliance control, our work is a novelty and a great contribution in this direction. Moreover, testing was carried out in clean rooms and in sterile production, where the up-to-date data on the appropriateness of the microbiological parameters of the working environment have an even greater significance. It was assumed that the bioluminescence method, despite the low microbial load of the controlled premices due to its working principle, is sufficiently sensitive to routine use and, due to simplicity, is useful as a quick and effective quality control tool and a tool for detecting changes and monitoring trends. Based on a six-year monitoring program of the microbiological conditions of sterile production at the UKCL Pharmacy, we prepared a microbiota catalog, which was used primarily as a basis for studying the suitability of the bioluminescence method for controlling the suitability of the working environment.
There was no significant difference between isolated species in a single clean room. In all, more than 70% of all isolated microorganisms human were their source. Among the isolates, there were 76.7% Gram-positive bacteria, 8.7% Gram-negative bacteria and 8.2% fungi and molds. The most commonly isolated were the bacteria of the genus Staphylococcus spp., Micrococcus luteus, Acinetobacter and sporogenic Bacillus spp.
The first and second hypotheses that the bioluminescence method of measuring the entire ATP is suitable for assessing the purity of the production environment and also suitable for validation and purification control, was confirmed by comparison with conventional microbiological methods. The third hypothesis that the method is sufficiently sensitive for use in clean rooms was confirmed by the fact that the quantities of ATP present in clean rooms were taken samples in the measuring range of equipment. In order to confirm the fourth hypothesis that there is a correlation between the measured quantity of RLU (Relative Luminescent Units) and CFU (Colony Forming Units) for a particular environment, the bioluminescent testing program was performed parallel to microbiological control. In order to evaluate the results and set up the action limits, we first divided the spaces into several meaningful categories, defined the action limits, and then searched for the correlation of methods by individual categories. We initially sought a correlation between CFU and RLU, but we were not able to confirm. The results were then evaluated for relevance and compared the method with respect to performance in finding an inadequate working surface. In two categories of premises, the methods were comparable and in two the ATP method was superior. In rooms with more stringent requirements, we did not confirm characteristic differences between methods, while in rooms with less stringent requirements and non-sterile production; we find the new method more sensitive. With used bioluminescence method, we measured the amount of ATP from living cells, which is directly correlated to the CFU and ATP, which is released from the dead microorganisms and from used materials. The latter is an actual indicator of pollution, as a potential medium for microorganisms or a potential source of pyrogens, which is of even greater importance for sterile production than the intracellular or microbial ATP.
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