Validation of an alternative method for sterility control of parenteral nutrition mixtures

5 October 2022

J-C. Mabin, C. Tournus, C. Chaplain, A. Diallo, X. Deviot
Centre Hospitalier de Saint-Denis, France

Parenteral nutrition mixtures (PNM) are produced in aseptic environment. It is essential to guarantee the sterility of the finished product by performing sterility tests. The reference methods proposed by the European Pharmacopoeia are membrane filtration and direct sowing. BACT/ALERT®VIRTUO® (Biomérieux) is an alternative microbiological method that allows indirect detection of bacterial growth by detecting CO2 production, with faster results and increased sensitivity compared to the reference methods.

The aim here is to validate a rapid microbiological method with the BACT/ALERT®VIRTUO® in order to perform a sterility test in routine.

The incubation media used are aerobic (FA+) and anaerobic (FN+) blood cultures bottles. After ensuring the fertility of the incubation media, the applicability of the method was tested using a standard PNM bag used in the hospital. Calibrated strains of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Aspergillus brasiliensis, Candida albicans (Bioball Single Shot®) were used for the different steps. They were put in solution and diluted to 5 CFU/mL before use. A growth test was performed on each blood culture bottle. For each germ, three pairs of samples were inoculated with 1 mL of microorganism (MO) solution and 3 ml of PNM, a positive control pair with 1 mL of MO and 3 mL of water for injection preparation and a negative control pair with 4 mL of water for injection preparation. These were incubated in the BACT/ALERT®VIRTUO® for 5 days at 35°C. The positive bottles were sown on Cos and PVX agars plates, then read and identified with the MALDI-TOF Biotyper® after 24 hours of incubation in the proofer. For molds and yeasts, subcultures on Can2 agar plates were read 48h later.

For each germ, no growth was detected for the negative controls. Growth was observed in the positive controls demonstrating an absence of inhibitory effect of the PNM. BACT/ALERT®VIRTUO® detected growth in all samples with a delay varying from 15 hours to 3 days for slow growing MO. The MALDI-TOF Biotyper® and Can2 agar plates (for molds and yeasts) confirmed that the detected MO corresponded to those inoculated.

These results allow the validation of the BACT/ALERT®VIRTUO® method for microbiological quality control of finished products. This method will then be applied on a test production before starting the production and its use in routine.

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