Late sampling for automated culture to extend the platelet shelf life to 5 days in Germany.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) is still a major challenge in transfusion medicine. Different methodologic concepts and screening strategies have been developed and investigated concerning their usability. We evaluated the feasibility of BacT/ALERT automated culture (BacT/A, bioMérieux) with late sampling after 3 days at the earliest. STUDY DESIGN AND METHODS: Twenty-four bacterial strains isolated from PCs and six relevant strains from reference stocks were spiked into apheresis-derived PCs (10-60 colony-forming units [CFU]/bag). Sampling was performed after 3 days, and bacterial detection was investigated using the two detection methods (BacT/A and BactiFlow [BF], bioMérieux). The maximum time-to-result of BacT/A was set to less than 12 hours. RESULTS: All medium- or high-pathogenic strains are capable of proliferating to high titers, and 100% of contaminated samples were detected by BF and BacT/A (6 to ≤12 h incubation); lower detection rates of BacT/A were obtained within 6 hours of incubation (≤6 h: 76.2-93.4%). The majority of low-pathogenic isolates are also capable of growing in PCs (89.7%), showing a detection rate of 74.3% for BF versus 54.3% for BacT/A (6 to ≤12 h incubation). BacT/A failed to detect bacteria within 6 hours of incubation. Certainly, a small number of strains did not grow under PC storage conditions and were detectable by BacT/A only with increased detection times. CONCLUSIONS: Late sampling after 3 days at the earliest, combined with reduced BacT/A incubation following the negative-to-date concept, offer an alternative opportunity to extend the shelf life of PCs from 4 to 5 days in Germany. The sensitivity of BacT/A with late sampling is nearly comparable to BF; the time-to-result is considerably longer.

Strain-dependent interactions of Streptococcus gallolyticus subsp. gallolyticus with human blood cells.

BACKGROUND: Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) is the causative pathogen in up to 20% of streptococcal-induced infective endocarditis (IE) cases. However, the underlying mechanisms of pathogenesis in S. gallolyticus have not yet been solved. Pathogens causing IE need to employ virulent strategies to initiate and establish infections, such as escape the bloodstream, invade the host-cell, and persist intracellularly. In this study, we examined the induction of inflammation by different S. gallolyticus strains in relation to their survival in whole blood and cell culture models as well as their ability to induce platelet aggregation. Phagocytosis of these bacteria by macrophages, followed by intracellular survival, was also quantified. METHODS: In whole blood and THP-1 cell culture assays bacterial growth kinetics was determined by plating, followed by colony counting. Induction of interleukin (IL)-6 expression in whole blood of three healthy volunteers, caused by different strains, was quantified by ELISA. Gene expression of cytokines (IL1B, IL6 and IL8) was quantified by real-time PCR after stimulating THP-1 monocytes with bacteria. Induction of platelet aggregation was analyzed by light transmission aggregometry using the BORN method. A macrophage model was used to analyze phagocytosis of strains and their survival in macrophages within 48 h. RESULTS: Strains promoted IL-6 secretion in a time-dependent fashion. For example, DSM16831 induced IL-6 secretion in whole blood earlier than other isolates, and was eliminated in the whole blood of one volunteer, whereas UCN34 could grow. Platelet aggregation depended on the different isolates used and on the individual platelet donor. Two strains (AC1181 and 010672/01) induced cytokine gene expression in THP-1 monocytes only marginally, compared to other strains. The phagocytosis rate of S. gallolyticus isolates differed significantly, and the isolates UCN34 and BAA-2069 could persist for a considerable time in the phagocytes. CONCLUSION: The strain-dependent differences of S. gallolyticus isolates, observed during interaction with human blood cells, support the hypotheses that divergences in individual virulence factors determine a distinct pathogenicity of the isolates. These data constitute an additional step towards the elucidation of mechanisms in the complex, multifactorial pathogenesis of this IE pathogen.

Genetic variants in genes of the inflammatory response in association with infective endocarditis.

AIMS: Inflammation in infective endocarditis (IE) is a complex network including interactions of inflammatory cytokines and other components of host response. Certainly, any variation in this network could influence susceptibility or disease progression of IE. In this study, 14 single nucleotide variants (SNVs) in genes coding for interleukin-1ß, interleukin-6, interleukin-10, toll-like receptor-4, tumor necrosis factor-α, selectin E and intercellular adhesion molecule-1 were analyzed for an association with susceptibility to IE and correlated with disease-related laboratory parameters. Furthermore, the occurrence of SNVs was examined to elucidate pathogen-dependent associations. METHODS AND RESULTS: The distribution of SNVs was determined in IE-patients and healthy blood donors by RFLP analysis. White blood cells (WBC) were counted using flow cytometry, concentration of C-reactive protein and procalcitonin was measured immunologically. Interleukin-6 c.471+870G>A genotypes differed significantly between IE patients and controls. The frequency of the heterozygote genotype GA was considerably higher in the patient group (68.9% vs. 43.8%, Pc<0.0003). Interleukin-6 c.-237 minor allele frequency was increased in patients, although not statistically significant. Additionally, we detected a potential relation between interleukin-1ß c.315C>T and IE. Pathogen-dependent analysis showed no significantly associated subgroup in relation to IE susceptibility, but gave hints towards alterations regarding Enterococcus-caused IE cases. Patients with genotype selectin-E c.-19 GT tend to have higher preoperative WBC counts than patients with genotype GG. We further showed an association between two interleukin-1ß SNVs and laboratory biomarkers. CONCLUSION: This study shows genetic predispositions for the establishment of IE. Furthermore, correlation of SNVs with disease-related biomarkers suggests a role of genetic variants regarding the inflammatory response in IE.