Bacterial contamination rate of platelet components by primary culture: a systematic review and meta-analysis.

BACKGROUND: Platelets have the highest bacterial contamination risk of all blood components, and septic transfusion reactions remain a problem. A good estimate of contamination rates could provide information about residual risk and inform optimal testing strategies. We performed a systematic review and meta-analysis of platelet contamination rates by primary culture. STUDY DESIGN AND METHODS: A literature search in December 2019 identified articles on platelet contamination rates using primary culture. We used meta-analysis to estimate the overall rate of contamination and meta-regression to identify heterogeneity. We studied the following sources of heterogeneity: collection method, sample volume, positivity criteria, and study date. Contamination rate estimates were obtained for apheresis (AP), platelet rich plasma (PRP), and buffy coat (BC) collection methods. RESULTS: The search identified 6102 studies, and 22 were included for meta-analysis. Among these 22 studies, there were 21 AP cohorts (4,072,022 components), 4 PRP cohorts (138,869 components), and 15 BC cohorts (1,474,679 components). The overall mean contamination rate per 1000 components was 0.51 (95% CI: 0.38-0.67) including AP (0.23, 95% CI: 0.18-0.28), PRP, (0.38, 95% CI: 0.15-0.70), and BC (1.12, 95% CI: 0.51-1.96). There was considerable variability within each collection method. Sample volume, positivity criteria, and publication year were significant sources of heterogeneity. CONCLUSION: The bacterial contamination rate of platelets by primary culture is 1 in 1961. AP and PRP components showed a lower contamination rate than BC components. There is clinically significant between-study variability for each method. Larger sample volumes increased sensitivity, and bacterial contamination rates have decreased over time.

Optimizing the safety of intradiscal platelet-rich plasma: an in vitro study with Cutibacterium acnes.

Aim: The most common risk associated with intradiscal injection of platelet-rich plasma (PRP) is discitis with Cutibacterium acnes. It is hypothesized that antimicrobial activity of PRP can be enhanced through inclusion of leukocytes or antibiotics in the injectate. Materials & methods: Multiple PRP preparations of varying platelet and leukocyte counts were co-cultured with C. acnes with or without cefazolin, with viable bacterial colony counts being recovered at 0, 4, 24 and 48 hours post-inoculation. Results: A direct correlation between C. acnes recovery and granulocyte counts were observed. Conclusion: We observed the greatest antimicrobial activity with the leukocyte-rich, high platelet PRP preparation combined with an antibiotic in the injectate. However, cefazolin did not completely clear the bacteria in this assay.

The impact of refrigerated storage of UVC pathogen inactivated platelet concentrates on in vitro platelet quality parameters.

BACKGROUND AND OBJECTIVES: Refrigeration (cold-storage) of pathogen inactivated (PI) platelet components may increase the shelf-life and safety profile of platelet components, compared to conventional room-temperature (RT) storage. Whilst there is substantial knowledge regarding the impact of these individual treatments on platelets, the combined effect has not been assessed. MATERIALS AND METHODS: Using a pool-and-split study design, paired buffy-coat derived platelets in 70% platelet additive solution (SSP+; MacoPharma) were left untreated or PI-treated using the THERAFLEX UV-Platelets System (UVC; MacoPharma). Units from each pair were split and stored at room temperature (20-24°C) or cold-stored (2-6°C) to yield RT, cold, RT-UVC and cold-UVC study groups (n = 8 in each group). In vitro quality and function was tested over 9 days. RESULTS: Cold-storage of UVC-treated platelets reduced glycolytic metabolism (glucose consumption and lactate production) compared to RT-UVC units. Cold-UVC platelets demonstrated complete abrogation of HSR by day 5, increased externalisation of phosphatidylserine (annexin-V binding) and activation of the GPIIb/IIIa receptor (PAC-1 binding) above the levels observed with the individual treatments. Aggregation responses (ADP and collagen) were enhanced in the cold-UVC platelets compared to both RT groups, but this was primarily mediated by cold-storage. Haemostatic function, as measured using TEG, was similar between the groups. CONCLUSION: Cold-storage of UVC-treated platelets reduced PI-induced acceleration of glycolytic metabolism. However, combining cold-storage and UVC-treatment resulted in additional phenotypic changes compared to each treatment individually. Further work is required to understand the impact of these changes in clinical efficacy.

Biofilm-associated accumulation-associated protein (Aap): A contributing factor to the predominant growth of Staphylococcus epidermidis in platelet concentrates.

BACKGROUND AND OBJECTIVES: Staphylococcus epidermidis is a predominant contaminant of platelet concentrates (PCs), outcompeting other skin flora bacteria such as Staphylococcus capitis. The accumulation-associated protein (Aap), encoded by the aap gene, is involved in formation of bacterial aggregates (biofilms) in S. epidermidis and is absent in S. capitis. In this study, the role of S. epidermidis aap in enhancing biofilm formation and conferring an advantageous growth in PCs was investigated. MATERIALS AND METHODS: Biofilm formation assays of S. epidermidis 1457, S. epidermidis 1457∆aap, S. capitis 517 and S. capitis 517 carrying S. epidermidis aap (S. capitis 517/pRBaap) were performed in glucose-supplemented trypticase soy broth (TSBg) and PCs. Additionally, competition assays with paired cultures (1:1 ratio) of S. epidermidis and S. capitis strains were seeded in PCs, followed by determination of viable counts of each organism at the end of PC storage. RESULTS: Staphylococcus epidermidis aap had no effect on biofilm formation in TSBg. By contrast in PCs, S. epidermidis 1457 showed higher biofilm formation than S. epidermidis 1457∆aap (P < 0·05). Biofilm formation was also enhanced in S. capitis 517/pRBaap compared to S. capitis 517 (P = 0·054). Competition assays showed that S. epidermidis 1457 outcompeted S. capitis 517, and importantly, S. capitis 517/pRBaap outcompeted S. capitis 517 and S. epidermidis 1457∆aap. CONCLUSION: This study demonstrated that S. epidermidis aap plays a role in biofilm formation in PCs conferring an advantageous proliferation to skin flora bacteria in this milieu. The molecular mechanisms of action of Aap merit further investigation.

Frequency of bacterial contamination in platelet concentrates in a tertiary care cardiac hospital in Rawalpindi, Pakistan.

Bacterial contamination of platelets concentrates (PCs) can result in transfusion transmissible infection. Storage temperature for platelets provides favourable environment for the bacterial growth. This study was conducted at Rawalpindi Institute of Cardiology, Rawalpindi, Pakistan from May, 2016 to July 2016. A total of 200 (48 hours stored) whole blood derived PCs collected were selected for the study. Sample were inoculated into Oxoid Signal blood culture bottles and incubated at 36±1°C for 07 days. Signal culture bottle with positive signals and visual appearance of turbidity were sub-cultured. Bacterial growth identification was carried out by standard reference methods. Out of 200 platelets concentrates, 63 suspected turbid and 02 with positive signal culture device were sub-cultured and identified. Staphylococcus aureus was identified in 02 bottles. The overall frequency of bacterial contamination in PCs was found to be 1%. The frequency of bacterial contamination in PCs found is very high as compared to developed counties. There is need of strict adherence to standard protocols for the prevention, early detection, and reporting of bacterial contamination in the PCs in Pakistan.

Bacterial survival and distribution during buffy coat platelet production.

BACKGROUND AND OBJECTIVES: At Canadian Blood Services, buffy coat (BC) platelet concentrates (BC-PCs) show a generally lower bacterial contamination rate than apheresis PCs. This study investigated whether the PC production method contributes to this observation. MATERIALS AND METHODS: Whole blood (WB) inoculated with eight bacterial strains was processed using the BC method. Bacteria were enumerated throughout BC-PC production and subsequent PC storage. Endotoxin production and bacterial adhesion to PC bags were evaluated during PC storage. PC quality was monitored by CD62P expression (flow cytometry) and changes in dynamic light scattering (ThromboLUX® ). RESULTS: During overnight WB hold, Staphylococcus epidermidis titres remained unchanged, commercial Escherichia coli and Klebsiella pneumoniae were eliminated and the remaining organisms proliferated to high concentrations. Through BC-PC production, bacteria segregated preferentially towards the cellular fractions compared to plasma (P < 0·05). During PC storage, most bacteria adhered to the PC bags and Gram negatives produced clinically significant endotoxin levels. Changes in CD62P expression or ThromboLUX scoring did not consistently reflect bacterial contamination in BC-PCs. CONCLUSION: WB hold during BC-PC production does not have a broad-spectrum bactericidal effect, and therefore, other factors contribute to low rates of contamination in BC-PCs.

Leukocyte presence does not increase microbicidal activity of Platelet-rich Plasma in vitro.

BACKGROUND: Human platelets are a rich reservoir of molecules that promote regenerative processes and microbicidal activity. This activity might be increased by concentration in platelet-rich plasma (PRP) products and modulated by the presence of leukocytes. Despite extensive use in clinical procedures, only few studies have investigated PRP's real microbicidal potential. Therefore, this study aimed at comparing the in vitro microbicidal activity of platelets and leukocyte-enriched PRP (L-PRP) to pure platelet-rich plasma (P-PRP) and the contribution of leukocytes to microbicidal properties. Antimicrobial effects of P- and L-PRP were tested against Escherichia Coli, Staphylococcus Aureus, Klebsiella Pneumoniae, Pseudomonas Aeruginosa and Enterococcus Faecalis. Furthermore, L-PRP was frozen (L-PRP cryo) to assess whether the preparation maintained in vitro characteristics. Microbicidal proteins released by the three preparations were also evaluated. RESULTS: L-PRP, L-PRP cryo and P-PRP generally induced comparable bacterial growth inhibition for up to 4 h' incubation, range 1-4 log. MIP-1α, RANTES, GRO-α, IL-8, NAP-2, SDF-1α and IL-6 showed strong microbicidal potential. CONCLUSIONS: We found in vitro antibacterial activity of L-PRP and P-PRP and the possibility to cryopreserve L-PRP, without important changes to its effectiveness; similar microbicidal activity between preparations containing or not leukocytes; and the contribution of three new molecules (NAP-2, SDF-1α and IL-6).

Platelet-rich plasma affects bacterial growth in vitro.

BACKGROUND AIMS: Platelet-rich plasma (PRP), a blood derivative rich in platelets, is a relatively new technique used in tissue regeneration and engineering. The increased quantity of platelets makes this formulation of considerable value for their role in tissue healing and microbicidal activity. This activity was investigated against five of the most important strains involved in nosocomial infections (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus faecalis) to understand the prophylactic role of pure (P)-PRP. Microbicidal proteins released from activated P-PRP platelets were also determined. METHODS: The microbicidal activity of P-PRP and platelet-poor plasma (PPP) was evaluated on different concentrations of the five bacterial strains incubated for 1, 2, 4 and 18 h and plated on agar for 18-24 h. P-PRP and PPP-released microbicidal proteins were evaluated by means of multiplex bead-based immunoassays. RESULTS: P-PRP and PPP inhibited bacterial growth for up to 2 h of incubation. The effect of P-PRP was significantly higher than that of PPP, mainly at the low seeding concentrations and/or shorter incubation times, depending on the bacterial strain. Chemokine (C-C motif) ligand-3, chemokine (C-C motif) ligand-5 and chemokine (C-X-C motif) ligand-1 were the molecules mostly related to Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis inhibition. Escherichia coli and Klebsiella pneumoniae were less influenced. CONCLUSIONS: The present results show that P-PRP might supply an early protection against bacterial contaminations during surgical interventions because the inhibitory activity is already evident from the first hour of treatment, which suggests that physiological molecules supplied in loco might be important in the time frame needed for the activation of the innate immune response.

Pathogen inactivation efficacy of Mirasol PRT System and Intercept Blood System for non-leucoreduced platelet-rich plasma-derived platelets suspended in plasma.

BACKGROUND AND OBJECTIVES: This study was conducted to evaluate the efficacy of pathogen inactivation (PI) in non-leucoreduced platelet-rich plasma-derived platelets suspended in plasma using the Mirasol PRT System and the Intercept Blood System. METHODS: Platelets were pooled using the Acrodose PL system and separated into two aliquots for Mirasol and Intercept treatment. Four replicates of each viral strain were used for the evaluation. For bacteria, both low-titre (45-152 CFU/unit) inoculation and high-titre (7·34-10·18 log CFU/unit) inoculation with two replicates for each bacterial strain were used. Platelets with non-detectable bacterial growth and platelets inoculated with a low titre were stored for 5 days, and culture was performed with the BacT/ALERT system. RESULTS: The inactivation efficacy expressed as log reduction for Mirasol and Intercept systems for viruses was as follows: human immunodeficiency virus 1, ≥4·19 vs. ≥4·23; bovine viral diarrhoea virus, 1·83 vs. ≥6·03; pseudorabies virus, 2·73 vs. ≥5·20; hepatitis A virus, 0·62 vs. 0·76; and porcine parvovirus, 0·28 vs. 0·38. The inactivation efficacy for bacteria was as follows: Escherichia coli, 5·45 vs. ≥9·22; Staphylococcus aureus, 4·26 vs. ≥10·11; and Bacillus subtilis, 5·09 vs. ≥7·74. Postinactivation bacterial growth in platelets inoculated with a low titre of S. aureus or B. subtilis was detected only with Mirasol. CONCLUSION: Pathogen inactivation efficacy of Intercept for enveloped viruses was found to be satisfactory. Mirasol showed satisfactory inactivation efficacy for HIV-1 only. The two selected non-enveloped viruses were not inactivated by both systems. Inactivation efficacy of Intercept was more robust for all bacteria tested at high or low titres.

Rapid detection of bacterial contamination of platelet-rich plasma-derived platelet concentrates using flow cytometry.

Flow cytometry can be used to detect bacterial contamination of platelet products. In this study, we investigated whether the incubation of a minimal volume of platelet-rich plasma (PRP)-derived platelet concentrates (PCs) with growth medium improved the analytical sensitivity of flow cytometry. Five bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli) were used. Platelets were inoculated with 10, 10(2), and 10(3) CFUs per mL; 0.5 mL, 1.0 mL, and 2.0 mL aliquots of spiked platelets were incubated with growth medium at 37°C for 24 hours. During the incubation period, the numbers of events were analyzed every 4 hours by flow cytometry. We could detect a low concentration (10 CFUs per mL) of bacteria in a small volume (minimum 0.5 mL) of PCs. Irrespective of spiking concentrations and incubation volumes, the detection times of S. aureus and S. epidermidis were 24 hours or less, while those of B. cereus, K. pneumoniae, and E. coli were 16 hours or less. A higher spiking concentration made it possible to shorten the detection time. The numbers of detected bacteria increased during the incubation. However, the graphs corresponding to K. pneumoniae and E. coli showed peak levels and decreasing patterns during the incubation period. The incubation of small volumes of PC with growth medium increased the analytical sensitivity of flow cytometry for bacterial detection. Therefore, flow cytometry can serve as a useful method for sterility testing using PRP-derived PCs with only low levels of consequent platelet loss.

Effect of Streptococcus sanguinis/Porphyromonas gingivalis single and combined biofilms upon platelet aggregation.

OBJECTIVE: To assess the effect of two oral bacteria Streptococcus sanguinis and Porphyromonas gingivalis upon platelet aggregation. MATERIALS AND METHODS: Streptococcus sanguinis, P. gingivalis, S. sanguniis+P. gingivalis were added to platelet-rich plasma and platelet aggregation measured using a platelet aggregometer. Platelets were passed through a flow chamber with S. sanguinis, P. gingivalis or a biofilm of S. sanguinis and P. gingivalis coated with saliva. Platelet adhesion to the chamber was observed under a fluorescence microscope for 15min. The positive control was platelets treated with adrenaline; the negative control was platelets treated with phosphate-buffered saline. RESULTS: The mean (± s.e.) aggregation magnitude of S. sanguinis and P. gingivalis was 77.7±7.4% and 79.3±9.9%, respectively. The aggregation magnitude of S. sanguinis+P. gingivalis was 51.3±12.9%, which was significantly lower than that for S. sanguinis/P. gingivalis (P<0.05). In the flow chamber system, platelets adhered to S. sanguinis/P.gingivalis respectively within 3min, and reached a plateau at 5-15min. Under the condition of the S. sanguinis- and P. gingivalis-saliva biofilm, platelet adhesion to the biofilm was significantly reduced at 5-15min (P<0.05). CONCLUSIONS: In the static or dynamic flow system, platelets adhered to S. sanguinis or P. gingivalis. However, if S. sanguinis was mixed with P. gingivalis, the aggregation magnitude (%) was significantly reduced.

Defect of glucosyltransferases reduces platelet aggregation activity of Streptococcus mutans: analysis of clinical strains isolated from oral cavities.

OBJECTIVE: Streptococcus mutans is a major pathogen of dental caries and occasionally isolated from the blood of patients with infective endocarditis, though the association of its cell-surface glucosyltransferases (GTFB, GTFC, and GTFD) with pathogenicity for infective endocarditis remains to be elucidated. In this study, we investigated the contribution of S. mutans GTFs to platelet aggregation and analysed GTF expression profiles in a large number of clinical oral isolates. DESIGN: The platelet aggregation properties of GTF-defective isogenic mutant strains constructed from S. mutans reference strain MT8148 were evaluated using whole blood and platelet-rich plasma (PRP) taken from mice, as well as human PRP. In addition, GTF expression profiles for 396 S. mutans strains isolated from the oral cavities of 396 subjects were analysed by western blotting using antisera specific for each GTF. RESULTS: The platelet aggregation activities of the GTF-defective isogenic mutants were significantly lower than that of MT8148 when added to a large number of cells. Western blotting revealed no strains without GTF expression, though six strains had alterations of GTFB and GTFC as compared to MT8148. PCR analyses indicated that the gtfB-gtfC region length was approximately 4.5 kb shorter in those strains as compared to MT8148. These were designated as "GTFBC-fusion" strains and they demonstrated lower levels of platelet aggregation. CONCLUSIONS: Our findings indicate that GTFs are associated with platelet aggregation. Although the clinical detection frequency of S. mutans strains with altered expressions is extremely low, GTFBC-fusion strains have activities similar to GTF-defective mutant strains.

Assessment of biofilm-forming ability of coagulase-negative staphylococci isolated from contaminated platelet preparations in Canada.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet (PLT) preparations and have been implicated in adverse transfusion reactions worldwide. The most frequently identified contaminant is Staphylococcus epidermidis, which is noted for its ability to maintain chronic hospital-acquired infections by forming biofilms as a chief virulence mechanism. STUDY DESIGN AND METHODS: Strains of S. epidermidis isolated from contaminated PLT preparations in Canada were distinguished via gene-specific polymerase chain reaction (PCR) with divIVA as a marker. Biofilm-forming ability was assessed by the presence of the gene icaD, slime production on Congo red agar, and biofilm formation on polystyrene surfaces. Production of polysaccharide intercellular adhesin (PIA) was resolved by immunofluorescence. RESULTS: Eight of the 13 (62%) CoNS isolates under study were identified as S. epidermidis. Of these, four strains (50%) were classified as strong biofilm producers. Three of the four biofilm-positive strains (75%) produced slime, harbored the icaD gene, and had positive expression of PIA. CONCLUSIONS: Despite the presumable commensal origin of the CoNS isolates, a large proportion of S. epidermidis strains demonstrated a potential for enhanced virulence. Identification of contaminant staphylococci as biofilm producers is thus relevant and informative with regard to treatment approach in the circumstance of inadvertent infection of a PLT recipient.

pH value promotes growth of Staphylococcus epidermidis in platelet concentrates.

BACKGROUND: The platelet (PLT) storage lesion is characterized metabolically by a pH value associated with lactic acid generation. PLT storage conditions support the growth of Staphylococcus epidermidis, the most common organism implicated in bacterial contamination of PLT concentrates (PCs). Here, different factors that influence bacterial growth in PCs are discussed and the relation between pH values of PCs and citrate plasma (CP) is studied, with emphasis on bacterial proliferation. STUDY DESIGN AND METHODS: The PLT lesion with regard to pH decrease and lactic acid production was monitored during storage and correlated to bacterial proliferation properties. A total of 115 coagulase-negative staphylococci, especially S. epidermidis isolates, were characterized for their proliferation in different blood components (CP, buffy coat-derived, and apheresis PCs). Furthermore, the influence of donor-specific, product-specific, species-specific, and strain-specific factors on bacterial proliferation was investigated. RESULTS: PCs showed a lower pH value in comparison to plasma during storage. Bacterial proliferation in PCs and the failure to grow in CP were determined with all organisms tested. No correlation to donor-specific, species-specific, or strain-specific factors was observed. Lowering the pH of CP resulted in bacterial proliferation, whereas a pH increase in the PC unit inhibited the proliferation of S. epidermidis. CONCLUSION: With emphasis on bacterial proliferation, the significant difference between PC and CP is the presence of metabolizing PLTs. The pH values of stored PLTs, but not those of stored plasma, support the growth of S. epidermidis.