Proof of concept for detection of staphylococcal enterotoxins in platelet concentrates as a novel safety mitigation strategy.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus is a predominant contaminant of platelet concentrates (PCs) that can evade detection during screening with culture methods. Importantly, S. aureus produces staphylococcal enterotoxins (SEs) during PC storage, which are linked to slow growth and enhanced biofilm formation. This study investigated timing of SE production during PC storage and feasibility of SE detection as a PC safety strategy. MATERIALS AND METHODS: Genomic and transcriptomic data of transfusion-relevant S. aureus PS/BAC/169/17/W, PS/BAC/317/16/W, CI/BAC/25/13/W and CBS2016-05 were used to determine the presence and differential expression of exotoxin genes in PCs. Trypticase soy broth (TSB) and PCs were inoculated with 1.0E+06 cfu/mL of S. aureus PS/BAC/169/17/W and CBS2016-05. Expression of SEs at different growth phases was confirmed with Western blotting. PCs were inoculated with 30 cfu/unit of the same strains, and SE detection during PC storage was optimized with a sandwich dot-ELISA assay. RESULTS: S. aureus genomes contain multiple exotoxin genes including those encoding for SEs. Transcriptome data revealed significant upregulation (0.5-6.7-fold, p < 0.05) of SE genes in PCs versus TSB. Western blots demonstrated SE production at all growth phases. Notably, dot-ELISA detected clinically relevant concentrations of SEs (~0.2 µg/mL) at 32 h of PC storage when S. aureus PS/BAC/169/17/W and CBS2016-05 counts were 1.8E+04 and 1.4E+04 cfu/mL, respectively. CONCLUSION: Genomic analyses revealed that staphylococcal exotoxins are widely distributed and highly conserved among transfusion-relevant S. aureus isolates. Furthermore, SEs are significantly upregulated in PCs and detected at 30 h of PC storage. Therefore, bacterial toxin detection could supplement mitigation strategies to enhance PC safety.

Challenging the 30-min rule for thawed plasma.

BACKGROUND AND OBJECTIVES: Frozen plasma (FP) is thawed prior to transfusion and stored for ≤5 days at 1-6°C. The effect of temperature excursions on the quality and safety of thawed plasma during 5-day storage was determined. MATERIALS AND METHODS: Four plasma units were pooled, split and stored at ≤-18°C for ≤90 days. Test units T30 and T60 were exposed to 20-24°C (room temperature [RT]) for 30 or 60 min, respectively, on days 0 and 2 of storage. Negative and positive control units remained refrigerated or at RT for 5 days, respectively. On Day 5, test units were exposed once to RT for 5 h. Quality assays included stability of coagulation factors FV, FVII, FVIII, fibrinogen and prothrombin time. Bacterial growth was performed in units inoculated with ~1 CFU/ml or ~100 CFU/ml of Serratia liquefaciens, Pseudomonas putida, Pseudomonas aeruginosa or Staphylococcus epidermidis on Day 0. RESULTS: Testing results of all quality parameters were comparable between T30 and T60 units (p < 0.05). Serratia liquefaciens proliferated in cold-stored plasma, while P. putida showed variable viability. Serratia epidermidis and P. aeruginosa survived but did not grow in cold-stored plasma. Positive and negative controls showed expected results. Overall, no statistical differences in bacterial concentration between T30 and T60 units were observed (p < 0.05). CONCLUSION: Multiple RT exposures for 30 or 60 min do not affect the stability of coagulation factors or promote bacterial growth in thawed plasma stored for 5 days. It is therefore safe to expose thawed plasma to uncontrolled temperatures for limited periods of 60 min.

Complete Genome Sequence of Staphylococcus aureus CI/BAC/25/13/W, Isolated from Contaminated Platelet Concentrates in England.

We present the genome sequence of Staphylococcus aureus CI/BAC/25/13/W, which was isolated in 2013 as a contaminant of a platelet concentrate with abnormal clotting at the National Health Service Blood and Transplant. Assessment of the genome sequence showed the presence of one chromosome (2,719,347 bp) and one plasmid (1,533 bp).

Complete Genome Sequence of Staphylococcus aureus PS/BAC/169/17/W, Isolated from a Contaminated Platelet Concentrate in England.

We report the genome sequence of Staphylococcus aureus PS/BAC/169/17/W, which was isolated in 2017 from a contaminated platelet concentrate at the National Health Service Blood and Transplant. Assessment of the genome sequence of this strain showed the presence of a 2,753,746-bp chromosome and a plasmid of 2,762 bp.

Genome Sequence of Staphylococcus aureus Strain PS/BAC/317/16/W, Isolated from Contaminated Platelet Concentrates in England.

We present the genome sequence of Staphylococcus aureus strain PS/BAC/317/16/W, which was isolated from contaminated platelet concentrates by the National Health Service Blood and Transplant in England (2017). Genome sequence analysis revealed the presence of one chromosome (2,665,983 bp) and two plasmids (4,265 bp and 2,921 bp) in this strain.

Assessing the inactivation capabilities of two commercially available platelet component pathogen inactivation systems: effectiveness at end of shelf life.

BACKGROUND AND OBJECTIVES: The inactivation capabilities of the two current commercially available pathogen inactivation (PI) systems for platelet components (PC), Mirasol and Intercept, were investigated by determination of the absence of viable bacteria at the end of shelf life by testing the entire contents of the PC by enrichment culture (terminal sterility). METHODS: A pool-and-split method was used, with two treated units and one untreated control per inoculum concentration. Pairs of PC bags were inoculated with a single bacterial species. Three concentrations (n = 2 per concentration), which incremented tenfold, were tested initially based on published data from the manufacturer. Dependent on these results, the concentrations subsequently tested were either increased or decreased until the inactivation capability of the system was derived. Bacterial count was determined post-spiking, immediately prior to treatment (2 h from spiking), immediately after treatment and at the end of shelf life (day seven). Enrichment culture was performed immediately prior to treatment, after treatment and at the end of shelf life. RESULTS: The inactivation capabilities, in CFU/ml, of Intercept and Mirasol, respectively, at the end of PC shelf life were as follows: Staphylococcus aureus ≥ 107 , <101 ; Staphylococcus epidermidis ≥106 , <102 ; Klebsiella pneumoniae 105 , <101 ; Streptococcus bovis ≥107 , 101 , Escherichia coli ≥106 , <101 ; Streptococcus pneumoniae ≥106 , 103 ; Streptococcus mitis ≥107 , 101 ; Listeria monocytogenes ≥107 , 101 ; Streptococcus dysgalactiae ≥107 , <101 ; Serratia marcescens 103 , <101 ; Pseudomonas aeruginosa 103 , Mirasol not tested; and Bacillus cereus < 102 , Mirasol not tested. CONCLUSION: The inactivation capability of Intercept was greater than that of Mirasol. Inactivation capability (by terminal sterility) is the most meaningful measure to evaluate a PI system for bacteria, rather than logarithmic reduction assessed immediately after treatment by plate count. PI offers a possible alternative to bacterial screening if treatment is performed at an appropriate time dependent on the inactivation capabilities of the system.

Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.