Validation of an ultra-fast microbiological control method for hospital preparations by scanning cytometry

Introduction

Hospital preparations (HP) for injection or instillation require sterility tests defined in the European Pharmacopoeia 8th Edition (2.6.1): either by membrane filtration, or by direct inoculation followed by 14 days of incubation. Quick release obligations for clinical use and short deadlines of some HP require rapid and sensitive sterility methods.

Objective

The main objective of this study was to achieve a sterility test validation with a scanning cytometry (SC), after filtration of the hospital preparation on a membrane. The specific objective was to confirm the applicability of this test for microbiological control in less than 48 hours.

Materials and methods

The ChemScan®RDI (bioMerieux) is an ultrasensitive microbial detection technology which could confirm the presence of a microorganism living in a liquid sample. After a membrane filtration (Ø 25mm, porosity 0.4ìm), the incorporation of a vital fluorochrome elicits a bacterial fluorescence which is analysed by the laser scanning (ëcx: 488 nm). The test of applicability was conducted on two antibiotics eyedrops and one bevacizumab solution for intracamelar injection (25 mg/ml), by membrane filtration (1 mL) followed by a controlled contamination (50 CFU) by S. aureus, B. subtilis, P. aeruginosa, C. sporogenes, C. albicans or A. brasiliensis, and the incorporation of the fluorochrome. The detection and the microbial enumeration were performed automatically, and then visually confirmed by optical microscopy by the operator.

Results

The quantitative detection of microbiological culture after 48h by SC showed the same cell viability ( 50 CFU/membrane) as the inoculum ( 50 CFU/mL), which was confirmed by visual inspection. However, the sample filterability (e.g. bevacizumab) significantly influenced the test applicability and required a rigorous adjustment of the test realization.

Conclusion
At the outset, we demonstrated that the SC is potentially applicable to rapid technical controls of HP sterility.