Bacteria In Plasma Research Articles

Verification of a method using magnetic bead enrichment and nucleic acid extraction to improve the molecular detection of bacterial contamination in blood components.

Bacterial contamination of blood products poses a significant risk in transfusion medicine. Platelets are particularly vulnerable to bacterial growth because they must be stored at room temperature with constant agitation for >5 days. The limitations of bacterial detection using conventional methods, such as blood cultures and lateral flow assays, include the long detection times, low sensitivity, and the requirement for substantial volumes of blood components. To address these limitations, we assessed the performance of a bacterial enrichment technique using antibiotic-conjugated magnetic nanobeads (AcMNBs) and real-time PCR for the detection of bacterial contamination in plasma. AcMNBs successfully captured >80% of four bacterial strains, including Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae, in both plasma and phosphate-buffered saline. After 24-h incubation with bacterial enrichment, S. aureus and B. cereus were each detected at 101 CFU/mL in all trials (5/5), E. coli at 101 CFU/mL in 1/5 trials, and K. pneumoniae at 10² CFU/mL in 4/5 trials. Additionally, without incubation, the improvement was also achieved in samples with bacterial enrichment, S. aureus at 10² CFU/mL and B. cereus at 101 CFU/mL in 1/5 trials each, E. coli at 10³ CFU/mL in 3/5 trials, and K. pneumoniae at 10¹ CFU/mL in 2/5 trials. Overall, the findings from this study strongly support the superiority of bacterial enrichment in detecting low-level bacterial contamination in plasma when employing AcMNBs and PCR.IMPORTANCEThe study presents a breakthrough approach to detect bacterial contamination in plasma, a critical concern in transfusion medicine. Traditional methods, such as blood cultures and lateral flow assays, are hampered by slow detection times, low sensitivity, and the need for large blood sample volumes. Our research introduces a novel technique using antibiotic-conjugated magnetic nanobeads combined with real-time PCR, enhancing the detection of bacteria in blood products, especially platelets. This method has shown exceptional efficiency in identifying even low levels of four different species of bacteria in plasma. The ability to detect bacterial contamination rapidly and accurately is vital for ensuring the safety of blood transfusions and can significantly reduce the risk of infections transmitted through blood products. This advancement is a pivotal step in improving patient outcomes and elevating the standards of care in transfusion medicine.

Probiotic Therapy During Vaccination Alters Antibody Response to Simian-Human Immunodeficiency Virus Infection But Not to Commensals.

The induction of robust circulating antibody titers is a key goal of HIV-1 vaccination. Probiotic supplementation is an established strategy to enhance microbiota and boost antibody responses to vaccines. A recent study tested whether oral probiotics could enhance vaccine-specific mucosal immunity by testing vaccination with and without supplementation in a Rhesus macaque Simian-Human Immunodeficiency Virus challenge model. Although supplementation was not associated with protection, the effects of probiotics on immunity after infection were not examined. To address this question, we measured antibody titers to HIV Env and commensal bacteria in plasma from the vaccination/supplementation time points as well as after Simian-Human Immunodeficiency Virus (SHIV) acquisition. We found that a trend toward lower HIV Env-specific titers in the animals given probiotics plus vaccine became greater after SHIV infection. Significantly lower Immunoglobulin (Ig) A titers were observed in animals vaccinated and supplemented compared with vaccine alone due to a delay in antibody kinetics at week 2 postinfection. We observed no difference, however, in titers to commensal bacteria during probiotic supplementation or after SHIV infection. These results suggest that probiotic supplementation may be a strategy for reducing IgA-specific HIV antibodies in the plasma, a correlate associated with increased HIV infection in the RV144 clinical trial.

Detection of Pathogenic Bacteria From Septic Patients Using 16S Ribosomal RNA Gene-Targeted Metagenomic Sequencing.

Conventional blood cultures were compared to plasma cell-free DNA-based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR)/next-generation sequencing (NGS) for detection and identification of potential pathogens in patients with sepsis. Plasma was prospectively collected from 60 adult patients with sepsis presenting to the Mayo Clinic (Minnesota) Emergency Department from March through August 2019. Results of routine clinical blood cultures were compared to those of 16S rRNA gene NGS. Nineteen (32%) subjects had positive blood cultures, of which 13 yielded gram-negative bacilli, 5 gram-positive cocci, and 1 both gram-negative bacilli and gram-positive cocci. 16S rRNA gene NGS findings were concordant in 11. For the remaining 8, 16S rRNA gene NGS results yielded discordant detections (n = 5) or were negative (n = 3). Interestingly, Clostridium species were additionally detected by 16S rRNA gene NGS in 3 of the 6 subjects with gastrointestinal sources of gram-negative bacteremia and none of the 3 subjects with urinary sources of gram-negative bacteremia. In the 41 remaining subjects, 16S rRNA gene NGS detected at least 1 potentially pathogenic organism in 17. In 15, the detected microorganism clinically correlated with the patient's syndrome. In 17 subjects with a clinically defined infectious syndrome, neither test was positive; in the remaining 7 subjects, a noninfectious cause of clinical presentation was identified. 16S rRNA gene NGS may be useful for detecting bacteria in plasma of septic patients. In some cases of gram-negative sepsis, it may be possible to pinpoint a gastrointestinal or urinary source of sepsis based on the profile of bacteria detected in plasma.

Potential of a Bacteriophage Isolated from Wastewater in Treatment of Lobar Pneumonia Infection Induced by Klebsiella pneumoniae in Mice.

The potential of bacteriophages as alternative treatment for multidrug-resistant (MDR) Klebsiella pneumoniae-related infections has recently gained much interest. The purpose of this research was to isolate and characterize a K. pneumoniae-specific lytic phage with the potential to treat experimental lobar pneumonia induced by K. pneumoniae in mice. A lytic phage was isolated from an urban wastewater sample in Tehran and characterized by transmission electron microscopy (TEM), thermal, pH, and chloroform stability before being employed for treatment of mice infected with K. pneumoniae in an experimental model of lobar pneumonia. BALB/C mice were challenged by intranasal inoculation with 108 colony-forming units (CFU/ml) of K. pneumoniae ATCC10031 followed by an intraperitoneal injection of the isolated phage using 1010 and 109 plaque-forming units (PFU/ml) simultaneously or 24h post infection. Control groups of mice received bacteria or bacteriophage alone. Mice were euthanized daily up to 7days post infection and examined for abnormality in their lungs and livers followed by determining the number of phages and bacteria in plasma and lung homogenates. The isolated phage (vB_KpnM-Teh.1) belonged to the Myoviridae family, was stable at 37°C, pH 7, and was resistant to chloroform. Treatment of mice with a single dose of phage simultaneously at the time of infection, or 24h post infection, resulted in seven and five logs decrease of CFU/ml in the lung homogenates up to 3days after phage administration, respectively. The isolated phage may have the potential as a therapeutic agent against K. pneumoniae infections.

Inactivation of bacteria in plasma by photosensitizers benzophenone and vitamins K3, B1 and B6 with UV A light irradiation.

A photosensitizer is a light-activated molecule that can generate reactive oxygen species or directly interact with nucleic acids. Both consequences can be applied to reduction of pathogens in various media and to selectively attack tumor cells. Numerous natural and synthesized photosensitizers have been identified for pathogen reduction. The photosensitizers of vitamins K3 (VK3), B1 (VB1), B6 (VB6) and benzophenone (BP) were prepared in 100-200 μM of PBS solution, irradiated with UVA at 0-48 J/cm2 for absorption spectrum alterations analysis. Bacteria species of E. coli, B. cereus, S. aureus and K. pneumoniae were mixed with 0-200 mM concentration of compounds and exposed to UVA irradiation of different dose at 6, 12 or 18 J/cm2 to assess the bactericidal effects. Over six logs CFU/ml reduction of E. coli suspended in PBS occurred after treatment with either VB1, VB6, VK3 or BP combined with UVA irradiation. When bacteria were suspended in plasma, two to seven logs reduction occurred depending on the UVA dose, photosensitizer concentration, and bacteria species. Among these photosensitizers, BP had the most potent bactericidal effect and is a promising UVA photosensitizer for pathogen reduction. The level of absorption spectrum alteration after UVA irradiation was profound for VK3 and VB6 but minimal for BP and VB1. The UV-vis absorption spectrum changes did not correlate with the bactericidal effect indicating that molecule modification by UVA light is not required for the bactericidal activity.

Factors affecting sedimentational separation of bacteria from blood.

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.

Key role of the number of complement receptor 1 on erythrocytes for binding of Escherichia coli to erythrocytes and for leukocyte phagocytosis and oxidative burst in human whole blood

AimTo study the role of complement receptor 1 (CR1) for binding of Escherichia coli (E. coli) to erythrocytes, for leukocyte phagocytosis, oxidative burst and complement activation in human whole blood from a CR1 deficient (CR1D) patient and healthy controls with low, medium and high CR1 numbers. MethodsAlexa-labelled bacteria were used to quantify erythrocyte-bound bacteria, free bacteria in plasma and phagocytosis using flow cytometry. Complement activation in plasma was measured by enzyme-linked immunosorbent assay. The CR1 numbers as well as C3bc and C4bc deposition on erythrocytes were measured by flow cytometry. Cytokines were measured using multiplex technology, and bacterial growth was measured by colony forming units. CR1 was blocked using the anti-CR1 blocking mAb 3D9. ResultsApproximately 85% of E. coli bound to erythrocytes after 15 min incubation in donor blood with high and medium CR1 numbers, 50% in the person with low CR1 numbers and virtually no detectable binding in the CR1D (r2 = 0.87, P < 0.0007). The number of free bacteria in plasma was inversely related to erythrocyte CR1 numbers (r2 = 0.98, P < 0.0001). E. coli-induced phagocytosis and oxidative burst were significantly enhanced by the anti-CR1 mAb 3D9 and in the CR1D and the donor with low CR1 numbers. E. coli-induced complement activation in plasma, C3bc and C4bc deposition on erythrocytes, and bacterial growth were similar in all four cases. ConclusionsCR1D and low CR1 numbers prevented E. coli binding to erythrocytes, increased free bacteria in plasma, phagocytosis and oxidative burst, but did not affect plasma or surface complement activation and bacterial growth.

The low photo-inactivation rate of bacteria in human plasma II. Inhibition of methylene blue bleaching in plasma and effective bacterial destruction by the addition of dilute acetic acid to human plasma.

Methylene blue (MB) and other photo-sensitizer molecules have been recognized as effective means for the inactivation of bacteria and other pathogens owing to their ability to photo-generate reactive oxygen species (ROS) including singlet oxygen. These reactive species react with the membrane of the bacteria causing their destruction. However, the efficiency of MB to destroy bacteria in plasma is very low because the MB 660 nm absorption band, that is responsible for the ROS generation, is bleached. The bleaching of MB, in plasma, is caused by the attachment of a hydrogen atom to the central ring nitrogen of MB, which destroys the ring conjugation and forms Leuco-MB which does not absorb in the 600 nm region. In this paper we show that addition of dilute acetic acid, ∼10(-4) M, to human plasma, prevents H-atom attachment to MB, allowing MB to absorb at 660 nm, generates singlet oxygen and thus inactivates bacteria. The mechanism proposed, for preventing MB bleaching in plasma, is based on the oxidation of cysteine to cystine, by reaction with added dilute acetic acid, thus eliminating the availability of the thiol hydrogen atom which attaches to the MB nitrogen. It is expected that the addition of acetic acid to plasma will be effective in the sterilization of plasma and killing of bacteria in wounds and burns.

Viable bacteria associated with red blood cells and plasma in freshly drawn blood donations.

ObjectivesInfection remains a leading cause of post-transfusion mortality and morbidity. Bacterial contamination is, however, detected in less than 0.1% of blood units tested. The aim of the study was to identify viable bacteria in standard blood-pack units, with particular focus on bacteria from the oral cavity, and to determine the distribution of bacteria revealed in plasma and in the red blood cell (RBC)-fraction.DesignCross-sectional study. Blood were separated into plasma and RBC-suspensions, which were incubated anaerobically or aerobically for 7 days on trypticase soy blood agar (TSA) or blue lactose plates. For identification colony PCR was performed using primers targeting 16S rDNA.SettingBlood donors attending Capital Region Blood Bank, Copenhagen University Hospital, Rigshospitalet, Hvidovre, Denmark, October 29th to December 10th 2013.Participants60 donors (≥50 years old), self-reported medically healthy.ResultsBacterial growth was observed on plates inoculated with plasma or RBCs from 62% of the blood donations. Growth was evident in 21 (35%) of 60 RBC-fractions and in 32 (53%) of 60 plasma-fractions versus 8 of 60 negative controls (p = 0.005 and p = 2.6x10-6, respectively). Propionibacterium acnes was found in 23% of the donations, and Staphylococcus epidermidis in 38%. The majority of bacteria identified in the present study were either facultative anaerobic (59.5%) or anaerobic (27.8%) species, which are not likely to be detected during current routine screening.ConclusionsViable bacteria are present in blood from donors self-reported as medically healthy, indicating that conventional test systems employed by blood banks insufficiently detect bacteria in plasma. Further investigation is needed to determine whether routine testing for anaerobic bacteria and testing of RBC-fractions for adherent bacteria should be recommended.

Rationale and mechanism for the low photoinactivation rate of bacteria in plasma

The rate of bacterial photoinactivation in plasma by methylene blue (MB), especially for Gram-negative bacteria, has been reported to be lower, by about an order of magnitude, than the rate of inactivation in PBS and water solutions. This low inactivation rate we attribute to the bleaching of the 660-nm absorption band of MB in plasma that results in low yields of MB triplet states and consequently low singlet oxygen generation. We have recorded the change of the MB 660-nm-band optical density in plasma, albumin, and cysteine solutions, as a function of time, after 661-nm excitation. The transient triplet spectra were recorded and the singlet oxygen generated in these solutions was determined by the rate of decrease in the intensity of the 399-nm absorption band of 9, 10-anthracene dipropionic acid. We attribute the bleaching of MB, low singlet oxygen yield, and consequently the low inactivation rate of bacteria in plasma to the attachment of a hydrogen atom, from the S-H group of cysteine, to the central nitrogen atom of MB and formation of cysteine dimer.

Inactivation of bacteria in plasma

The photo-inactivation rate of bacteria by methylene blue, MB, was found to be significantly lower in plasma than in water, saline, and PBS solutions. The spectroscopic data and ultrafast time resolved transient spectra and kinetics presented show that methylene blue photo-bleaches faster and to a larger degree in plasma and the MB excited singlet and triplet state populations in plasma are much lower in plasma than in water and PBS solutions. The optical density, OD, of MB in plasma was found to decrease by ~50% after a minute of illumination with 661 nm light, while under identical conditions the OD in PBS solution decreased by only 1%. Based on these data and the effect of the plasma proteins on MB photochemistry, a mechanism is proposed that accounts for the low inactivation rate of bacteria in plasma.

Combinatorial Antimicrobicidal Peptides Exhibit Enhanced Activity Against Bacterial Contaminants In Stored Platelet Concentrates (PCs).

AbstractAbstract 1124▪ Introduction:Contamination of blood and blood components by bacteria, virus, fungi and parasites is a major safety risk in transfusion medicine. Novel proof-of-concepts for microbial reduction that enhance risk-to-benefit ratio of the treated products are still an unmet public health need in transfusion medicine. We have recently shown that synthetic antimicrobicidal peptides (AMPs) originating from thrombin-induced human platelet-derived antimicrobial proteins (named PD1-PD4) and repeats of Arginine-Tryptophan (RW1-RW5), demonstrate microbicidal activity against bacteria in plasma and PCs. In the present study we selected RW2, RW3 and RW4 and tested the effect of the peptides individually and in various combinations (cocktail) to see whether the cocktail approach enhances the antimicrobicidal activity of these peptides. Methods:The RW2, RW3 and RW4 peptides were tested on platelet samples spiked with 104 colony forming units (cfu) of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa. Each spiked sample was incubated individually with 10 mM of RW2, RW3, and RW4 peptides for 1 hour at 37°C to observe their individual effect. Peptides were then combined in the following way to assess their cocktail effect: RW2+RW3; RW2+RW4; RW3+RW4; RW2+RW3+RW4. Spiked sample without any peptide was included as control. Following incubation with the peptide a ten-fold dilution of the inoculum was made and a fixed volume plated on nutrient agar plates. The plates were incubated at 37°C for 18–24 hours and colonies were counted and expressed as cfu/ml. Results:Our analysis revealed that individual peptides RW2, RW3 and RW4 were either ineffective or were moderately microbicidal (100-fold reduction) against S. aureus, S. epidermidis and P. aeruginosa. However, in combination (cocktail), these peptides together demonstrated enhanced microbicidal activity against bacteria in spiked platelets. The combinations, RW2+RW4, RW3+RW4 and RW2+RW3+RW4 were the most effective that resulted in 1000-fold reduction of all three bacteria tested. Conclusion:The present study conclusively demonstrates the synergistic effect of antimicrobicidal peptides whose combined microbicidal activity is 1000-fold higher than when the peptide is acting alone. By selecting appropriate combination of AMPs against specific bacteria of interest, new proof-of-concepts could be developed that are alternatives to the current pathogen reduction agents.The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Pharmacokinetics of enrofloxacin after single intravenous administration in sheep

The disposition of enrofloxacin in sheep was investigated after single-dose intravenous administration of 2.5 mg/kg body weight. Blood samples were drawn from the jugular vein at predetermined times after drug administration. Plasma concentrations of enrofloxacin and its active metabolite ciprofloxacin were simultaneously determined by reverse-phase high performance liquid chromatography. The data collected were subjected to non-compartmental and compartmental kinetic analysis. Statistical model theory was used to determine non-compartmental pharmacokinetic parameters. Disposition of enrofloxacin was described by a three-compartment open model with elimination from the central compartment following intravascular administration. The elimination half-life, the volume of distribution, and the area under the concentration vs time curve (AUC) were 4.31 h, 1.10 l/kg and 9.24 microg x h/ml, respectively. Enrofloxacin was metabolised to ciprofloxacin and the ratio between the AUCs of ciprofloxacin and enrofloxacin was 0.26 after intravenous administration. With predictive models of efficacy (maximum plasma concentrations/minimum inhibitory concentrations [Cmax/MIC] and AUC/MIC ratios in plasma) for most of the sheep pathogen microorganisms, enrofloxacin produced scores higher than 15 and 50, respectively. After intravenous administration atthe dose of 2.5 mg/kg, enrofloxacin achieved concentrations several times above the MIC for major pathogen bacteria in plasma, and it may prove useful in the treatment of infectious diseases caused by sensitive pathogens in sheep.

Phage-Displayed Peptide-Q Dot Nanocrystal Combo for High-Sensitivity Bacterial Detection in Plasma.

Abstract 3153Poster Board III-90 IntroductionDespite improved phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage, bacterial contamination of transfusion products is still a longstanding problem. Current bacterial detection tests relevant to transfusion medicine, especially for stored platelets have limitations with regard to time, specificity and sensitivity. There is a need for new and improved cost-effective high-affinity detection probes to fill this gap. In this study, using plasma spiked with Bacillus cereus 4342 and B. anthracis-Sterne as an experimental system, we identified peptides from a bacteriophage-displayed random peptide library that selectively bind and detect the Bacillus strains. By labeling the peptides with Q dot-liquid nanocrystals, the detection sensitivity of the peptides was further enhanced. MethodsA commercially available bacteriophage-displayed random peptide library was screened using B. cereus 4342 as bait, using appropriate controls under stringent conditions. The screening and subsequent sequencing of the phage DNA identified two phages each containing a coding sequence for 12-amino acid peptide that are selectively capable of binding to the Bacillus. Based on the nucleic acid sequence, the two synthetic peptides with biotin tag were prepared for detection assays and to enhance the detection sensitivity further, the peptides were labeled with streptavidin-conjugated fluorescent quantum-dots (Q dots). Fluorescence was measured either by a plate reader or using a fluorescence microscope. ResultsThe two synthetic peptides selectively bound to the Bacillus strains in both dot blot and ELISA assays. The membrane-based dot blot assay demonstrated an assay sensitivity of 103 colony forming units/ml (CFU/ml), whereas ELISA demonstrated a sensitivity of 102 CFU/ml detection limit. Fluorometry analysis of spiked plasma samples revealed that the two peptides were able to bind to B. cereus 4342 and B. anthracis Sterne and detect these bacteria in plasma at 102 CFU/ml concentrations. The peptide-Qdot combo even detected a single bacterium in fluorescence microscopy. ConclusionOverall, the results reported here validate the usefulness of affinity-selected recombinant filamentous phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity detection probes for bacteria in various platforms and settings relevant to the blood safety and transfusion medicine.The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. DisclosuresNo relevant conflicts of interest to declare.

Development of an internally controlled PCR assay for broad range detection of bacteria in platelet concentrates

A real-time PCR assay based on the 16S rRNA gene was optimized for the detection of a broad range of bacteria in plasma and platelet concentrates (PC). A lambda phage internal control was constructed and implemented in the assay, which made it suitable for diagnostic use. Spiking studies in plasma and PCs were performed to determine the analytical sensitivity of the assay. Thirty three colony forming units (CFU)/ml of E. coli and 72 CFU/ml of Staphylococcus epidermidis could be detected in plasma, and 97 CFU/ml of S. epidermidis in PCs. The assay detected all bacteria relevant for bacterial contamination of PCs. The short turn around time of the assay made it suitable for testing PCs for bacterial contamination prior to transfusion.

A Randomized, Double-Blind Clinical Trial of a 3-Week Course of Doxycycline plus Albendazole and Ivermectin for the Treatment of Wuchereria bancrofti Infection

Eight- and 6-week courses of doxycycline are superior to standard treatment of bancroftian filariasis. Standard treatment (albendazole plus ivermectin) is associated with adverse reactions. We assessed whether a shorter (i.e, 3-week) course of doxycycline with standard treatment would show superior efficacy to standard treatment alone and reduce the incidence of adverse reactions. A total of 44 adults from Ghana were recruited in January 2003: 20 received doxycycline (200 mg/day) for 3 weeks, and 24 received matching placebo. Participants received albendazole (400 mg) and ivermectin (150 microg/kg) at month 4, and adverse reactions were assessed 48 h later. Treatment efficacy was evaluated at months 4, 12, and 24. The microfilariae level was significantly reduced after receipt of doxycycline treatment at months 4 (P = .017), 12 (P = .001), and 24 (P = .005). The microfilariae level was only significantly reduced at month 12 in the placebo group (P = .041). At all follow-up points, the microfilariae level was significantly lower in the doxycycline group. Adverse reactions to standard antifilarial treatment were similar in frequency between the doxycycline group (in 7 of 11 subjects) and the placebo group (in 13 of 17 subjects). Moderate reactions only occurred in the placebo group (in 3 of 17 subjects). Severity of adverse reaction was associated with microfilaremia (P = .037), Wolbachia bacteria in plasma (P = .048), and proinflammatory cytokines in plasma (P = .019). Adult parasite viability was not significantly different between doxycycline and placebo groups at months 12 or 24. Treatment with doxycycline for 3 weeks is more effective in inducing a long-term amicrofilaremia than is standard treatment alone, but it is ineffective at inducing curative effects. Inflammatory reactions to antifilarial treatment are associated with levels of microfilariae and Wolbachia endosymbionts released into plasma.

Impact of manufacturing, irradiation and filtration steps to bacterial contamination of autologous fibrin sealant

Background Preoperative production of autologous fibrin sealant has become a routine procedure during the last years. As a certain percentage of blood products is contaminated with bacteria, contamination of plasma used for the production of fibrin sealant cannot be excluded. Especially in the orthopaedic setting, application of contaminated fibrin sealant can cause severe infections. Materials and methods We contaminated plasma with Staphylococcus epidermidis, Corynebacterium striatum, Bacillus subtilis or Escherichia coli and produced fibrin sealant by cryoprecipitation and alcohol precipitation. Additionally, the products were gamma-irradiated at a dose of 30 Gy, frozen at −55 °C and filtered through a 0.2 μm filter after thawing. After each preparation step, samples were drawn and numbers of colony forming units were counted after incubation on agar plates. Results Cryoprecipitation, irradiation, freezing at −55 °C, and alcohol precipitation have only little impact on numbers of colony forming units. Filtration through a bacterial filter results in a sterile product. Conclusion Bacteria in plasma as a starting material for production of fibrin sealant survive all routine steps of production, including gamma irradiation and freezing. Filtration of the product through a qualified bacterial filter is the only safe means to provide a sterile product.

Polymorphonuclear neutrophil chemotaxis under aerobic and anaerobic conditions

The motility of human polymorphonuclear neutrophils was studied in vitro under aerobic and anaerobic conditions. Chemotactic factors were generated from plasma with immune complexes or with whole bacteria (Staphylococcus aureus, Escherichia coli, and Bacteroides fragilis). Chemotaxis induced by chemotactic factors generated from immune complexes was identical under both conditions. However, chemotaxis utilizing chemotactic factors generated from bacteria was markedly depressed under anaerobic conditions. Mean random tubemoltility was not significantly different under aerobic and anaerobic conditions. These data indicate that different metabolic pathways may be involved in polymorphonuclear neutrophil movement. Some of these pathways require oxygen (chemotaxis in response to factors generated by bacteria in plasma), whereas others do not (random tube migration and chemotaxis in response to factors generated by immune complexes in plasma). These observations may be important in the induction of inflammatory responses within hypoxic tissues.

PRESERVATION OF LIQUID PLASMA BY SULFONAMIDE DERIVATIVES

To the Editor:— In the February 14 issue ofThe JournalI published a paper on the preservation of liquid plasma by the use of sulfonamide derivatives. The report was based on hundreds of experiments employing numerous species of bacteria encountered as contaminants in stored plasma. In no instance did I demonstrate growth of bacteria in plasma containing 200 mg. per hundred cubic centimeters (0.2 per cent) of sodium sulfathiazole, nor did plasma containing the compound become grossly contaminated when exposed to air contamination for long periods of time. InThe Journalof March 28, Heath and Province published their results on a small series of experiments carried out under unusual conditions in that they do not represent actual working conditions and made broad conclusions which do not discredit my own but tend to cast doubt on the efficacy of the sulfonamide method. In their experiments they used only five