Improvements of ATP Assay as a Substitute for the CFU Method in Estimating Viable Cell Count for BCG/rBCG Vaccine Preparations

Abstract

Bacillus Calmette-Guérin (BCG) and recombinant BCG (rBCG) vaccines can be genetically traced back to a live attenuated strain of Mycobacterium bovis. As organism viability is essential for the stimulation of a protective immune response, monitoring the count of viable organisms is an integral part of vaccine quality control. The colonyforming unit (CFU) test has been the conventional assay for determining BCG viability, and is a widely accepted surrogate for BCG potency. CFU analysis, however, is problematic and time consuming. The slowness and high variability of CFU test results are the main driving forces for manufacturers and control laboratories to look for a rapid, more reproducible alternative viable count assay. A modified adenosine triphosphate (ATP) luminescence assay was developed by Statens Serum Institut and was promoted by the WHO as an alternative viable count assay. However, certain conditions during the processes of sample preparation and ATP extraction have to be established before the ATP assay can meet the requirements of robustness and reproducibility. This study is focused on identifying the conditions necessary for a reliable process of ATP analysis for BCG/rBCG preparations. Using our improved ATP assay protocol, we demonstrated that the correlation coefficient between CFU count and ATP concentration of BCG/rBCG vaccines was high (R 2 =0.83 for accelerated stability samples and R 2 >0.97 for all other preparations). The ATP luminescence assay is a rapid, sensitive, reliable, strain-non-specific method in quantification of the viability of live attenuated mycobacterial vaccine preparations.

Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

“…In vitro viability was assessed using the colony forming unit (CFU) assay 64 . At indicated time points 100 µl of each culture was harvested, and 10-fold serial dilutions were made with Saline Tyloxapol Buffer (STB) to www.nature.com/scientificreports/ disrupt bacteria clots 65 before plating 10 µl on Middlebrook 7H10/OADC agar supplemented with appropriate antibiotics. The plates were then incubated at 37 °C for 3 weeks after which colonies were counted from dilutions that yielded 20-100 colonies and CFU/ml values calculated.…”

Development of a New Approach to Determine the Potency of Bacille Calmette–Guérin Vaccines Using Flow Cytometry

“…However, analyst variation of the CFU assay is high (5%–50%) owing to dilution error, modification of medium during cultivation, and counting error arising from mycobacterial clumping. In addition, 4 to 5 weeks are needed for colony formation because of a very slow growth rate, resulting in long lead-time during production of vaccines [ 3 , 4 ]. Accordingly, it is necessary to develop a new potency testing method to rapidly determine the viable cells in BCG vaccine.…”