Combining culture and microbead-based immunoassay for the early and generic detection of bacteria in platelet concentrates.

BACKGROUND: Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS: A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS: Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION: In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates.

A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10-102 CFU mL-1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL-1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

An innovative biologic recycling process of leukoreduction filters to produce active human antimicrobial peptides.

BACKGROUND: Human neutrophil peptides (HNPs) 1 to 3 are the major antimicrobial peptides of the azurophilic granules of neutrophils. They represent an important arm of the innate immune system. Their production by chemical synthesis and recombinant technologies is expensive and limited by technical constraints due to their composition and the presence of three disulfide bonds. STUDY DESIGN AND METHODS: We have developed an original approach based on the purification of the natural human defensins HNPs 1 to 3 from neutrophils trapped on leukoreduction filters used in blood processing. The purification of HNPs 1 to 3 from these filters is performed in two steps: extraction of HNPs 1 to 3 retained in the filters followed by their immunoprecipitation. Studies were performed to determine the stability of defensins in the filters stored at room temperature. The activity of HNPs 1 to 3 obtained by our rapid protocol was validated by determining minimal inhibitory concentrations (MICs) against six reference bacterial strains and 12 clinical isolates. RESULTS: The human defensins HNPs 1 to 3 extracted from leukoreduction filters displayed high antimicrobial activity against tested strains, with MIC values between 0.12 and 1 µg/mL. Kinetics assays showed the appearance of activity 15 minutes after peptide addition. Moreover, we found that the HNPs 1 to 3 purified from leukoreduction filters that had been stored for 45 days at room temperature remained active. CONCLUSION: Leukoreduction filters provide a rich and safe source of active human defensins HNPs 1 to 3. Moreover, the stability of the peptides in filters stored at room temperature allows envisaging a large-scale development of the process.

Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria.

Blood safety is a global health goal. In developed countries, bacterial contamination of platelet concentrates is the highest infectious risk in transfusion despite the current preventive strategies. We aimed to develop a conductometric biosensor for the generic, rapid and sensitive detection of Gram-negative bacteria. Our strategy is based on immunosensors: addressable magnetic nanoparticles coupled with anti-LPS antibodies were used for the generic capture of Gram-negative bacteria. Bacterial capture was characterized by impedancemetric and microscopic measurements. The results obtained with conductometric measurements allowed real-time, sensitive detection of Escherichia coli or Serratia marcescens cultures from 1 to 10(3) CFU mL(-1). The ability of the immunosensor to detect Gram negative bacteria was also tested on clinically relevant strains. The conductometric immunosensor allowed the direct detection of 10-10(3) CFU mL(-1) of Pseudomonas aeruginosa and Acinetobacter baumannii strains that were undetectable using standard immunoblot methods. Results showed that the conductometric response was not inhibited in 1% serum.