Diagnosis and Stratification of Pseudomonas aeruginosa Infected Patients by Immunochemical Quantitative Determination of Pyocyanin From Clinical Bacterial Isolates.

The development of a highly sensitive, specific, and reliable immunochemical assay to detect pyocyanin (PYO), one of the most important virulence factors (VFs) of Pseudomonas aeruginosa, is here reported. The assay uses a high-affinity monoclonal antibody (mAb; C.9.1.9.1.1.2.2.) raised against 1-hydroxyphenazine (1-OHphz) hapten derivatives (PC1; a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids). Selective screening using PYO and 1-OHphz on several cloning cycles allowed the selection of a clone able to detect PYO at low concentration levels. The microplate-based ELISA developed is able to achieve a limit of detection (LoD) of 0.07 nM, which is much lower than the concentrations reported to be found in clinical samples (130 µM in sputa and 2.8 µM in ear secretions). The ELISA has allowed the investigation of the release kinetics of PYO and 1-OHphz (the main metabolite of PYO) of clinical isolates obtained from P. aeruginosa-infected patients and cultured in Mueller-Hinton medium. Significant differences have been found between clinical isolates obtained from patients with an acute or a chronic infection (~6,000 nM vs. ~8 nM of PYO content, respectively) corroborated by the analysis of PYO/1-OHphz levels released by 37 clinical isolates obtained from infected patients at different stages. In all cases, the levels of 1-OHphz were much lower than those of PYO (at the highest levels 6,000 nM vs. 300 nM for PYO vs. 1-OHphz, respectively). The results found point to a real potential of PYO as a biomarker of P. aeruginosa infection and the possibility to use such VF also as a biomarker for patient stratification[2] and for an effective management of these kinds of infections.

Immunochemical Determination of Pyocyanin and 1-Hydroxyphenazine as Potential Biomarkers of Pseudomonas aeruginosa Infections.

A novel immunochemical approach to diagnose Pseudomonas aeruginosa infections is reported, which is based on the quantification of relevant and specific virulence factors secreted by this microorganism. Specific antibodies have been raised using hapten PC1 (a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids), designed to recognize 1-hydroxyphenazine (1-OHphz), which is the main metabolite of pyocyanin (PYO). PYO is one of the most important virulence factors produced by nearly all P. aeruginosa strains, and other species do not produce this factor. With these antibodies, an immunochemical analytical procedure able to quantify both 1-OHphz and PYO in complex clinical samples has been developed. 1-OHphz can be directly measured in solubilized sputum samples diluted 20 times with the assay buffer. Quantification of PYO is accomplished after conversion to 1-OHphz in just 20 min under basic conditions. A LOD of 0.60 ± 0.01 nM (4.80 ± 0.08 nmol kg(-1) sputum) is reached for both biomarker targets under the conditions established, a value that is much below the reported concentrations on sputum samples obtained from infected patients (up to 100 µM). The assay is robust, reproducible, accurate, can be run in about 2 h, and many samples can be measured simultaneously. The present reported assay could represent a significant improvement in the diagnosis of infectious diseases caused by this pathogen.

An immunochemical strategy based on peptidoglycan synthetic peptide epitopes to diagnose Staphylococcus aureus infections.

The characteristic pentaglycyl cross-bridge of the Staphylococcus aureus peptidoglycan (PG) cell wall component is an attractive epitope to raise specific antibodies against this microorganism. Based on this approach, we report here for the first time a competitive ELISA able to detect S. aureus down to 10(4) CFU mL(-1), without pre-enrichment on cell culture. The antibodies were raised against peptide-protein bioconjugates prepared by covalently coupling peptide haptens (PSau6 and PSau8) designed and synthesized taking into consideration the complex tridimensional structure in the PG polymer. Deglycosylation of the PG under acidic conditions has found to increase assay detectability. Assay performance has been evaluated in clinical samples such as bronchoalveolar lavage (BAL) and bronchoalveolar endotracheal aspirates (BAS) showing promising results for further implementation of this immunoassay as a daily routine diagnostic tool. Cross-reactivity studies have demonstrated that the immunoassay is specific for S. aureus.