Isolation of pathogenic bacteria from sputum samples using a 3D-printed cartridge system.

Within this contribution we introduce a 3D-printed cartridge system enabling the convenient and cost-efficient sample preparation from sputum for subsequent PCR based detection schemes. The developed fluidic system operates on pneumatic actuations. The closed system ensures a very low probability for contamination during sample processing, which is crucial when using a highly sensitive detection method such as PCR. The enrichment of the bacterial cells is achieved using different types of amine-functionalized particles. Our particle-based sample preparation approach yields intact and viable bacterial cells. Accordingly, not only PCR-based detection schemes can be employed, but also spectroscopic methods and biochemical tests, which require cultivation steps, are possible. The cartridge design in principle is compatible with magnetic and non-magnetic particle types. We investigated both variants and found that the performance of expanded glass beads is superior over the magnetic particles within the cartridge. Owing to the rather large size of the expanded glass beads, the dimensions of the channels can be enlarged, leading to lower hydrodynamic resistances, which is beneficial when processing viscous samples such as sputum. We verified the performance of our system using both artificial and real sputum samples containing Escherichia coli and Moraxella catarrhalis.

Loop-mediated amplification as promising on-site detection approach for Legionella pneumophila and Legionella spp.

Recently Legionella pneumophila is the main causative waterborne organism of severe respiratory infections. Additionally, other Legionella species are documented as human pathogens. In our work, we describe a rapid detection method which combines two advantages for sensitive and specific detection of the genus Legionella: the fast isothermal amplification method "Loop-mediated isothermal AMPlification" (LAMP), and a colorimetric detection method using the metal indicator hydroxynaphtol blue (HBN) which allows to determine an optical signal with a simple readout (with the naked eye). Moreover, we present two approaches for minimizing the assay volume using a stationary microchip LAMP and droplet digital-based LAMP (ddLAMP) as promising highly sensitive setups.

Rapid Isolation and Identification of Pneumonia-Associated Pathogens from Sputum Samples Combining an Innovative Sample Preparation Strategy and Array-Based Detection.

With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.