Potential Mitigation of the Covert Risk of Bacterial Contamination of RBC: Inactivation of Propionibacterium Acnes in RBC Using Amustaline and Glutathione

Background: Propionibacterium acnes is usually considered a commensal anaerobic non-pathogenic bacterium, which, originating from skin flora, is often found responsible for blood product contamination, including RBC. Various levels of risk are assigned to the different pathogens in RBC, with the bacterial risk of sepsis estimated to 1 in 2 million based on reported cases (Kleinman & Stassinopoulos, Transfusion 2015). RBC bacterial contamination is not assessed in routine practice and refrigerated storage is often given as the explanation for the lack of reported cases. In a recent study from the Canadian Blood Services (Kumaran D. et al ., Vox Sanguinis, 2017, 112(suppl),73) P. acnes was implicated in a severe transfusion reaction of a 59-year old patient suffering from acute leukemia. The patient recovered after antibiotic treatment, however this incident highlighted the risk associated with the transfusion of RBC components that are usually not tested for bacterial contamination. The same and related Propionibacterium- species were found to be contaminating 9/19 RBC components tested at outdate after day 43 of storage. Currently, only platelet components are routinely screened for the presence of bacteria in countries where bacterial culture screening has been adopted.

A system for the broad spectrum inactivation of pathogens in RBCs is under development using the nucleic acid targeting technology that employs amustaline (S-303) and glutathione (GSH). The RBC recovery of the S-303/GSH treated RBC has been found to meet the FDA criteria and robust inactivation of various pathogens has been demonstrated, including emerging and established pathogens. Inactivation of bacteria by the system continues to be investigated.

Methods and Results: In this study we used P. acnes acquired from the California State Health Department Services, identified after a case of sepsis and characterized through sequencing and MIDI analysis (CDHS 00A-6608). A highly concentrated bacterial stock (8.8 log₁₀/mL) was generated by propagation in thioglycolate medium. It was then diluted 1:100 to a final titer of 6.6 log/mL into an RBC pool of two matched units prepared in AS-1 , that was subsequently split into two identical RBC units. One unit remained untreated (Control), while the parallel unit (Test) was treated using S-303 and GSH for ~20 h before an "exchange" step consisting of centrifugation and resuspension of RBC in SAG-M additive solution. Samples were harvested at 3 h and 20 h post-exchange for the Test unit and at 3 h and 20 h post-incubation for the Control unit. Test samples were screened by quantitative plate culture under anaerobic condition, while the Control samples were serially diluted to countable concentrations before similar culture screening. While bacterial titers remained unchanged in the Control unit, no viable bacteria were detected in the Test unit post-treatment, indicating a >6.6 log₁₀/mL inactivation.

Conclusion: The results of this study show that the covert risk of bacterial contamination in RBC components could be addressed by the pathogen reduction technology using S-303/GSH, even when bacterial titers far exceed the ones expected in refrigerated RBC at the end of shelf life.