Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci.

BACKGROUND: VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. OBJECTIVES: To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. METHODS: NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. RESULTS: All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. CONCLUSIONS: NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

Improvement of the Immunochromatographic NG-Test Carba 5 Assay for the Detection of IMP Variants Previously Undetected.

Here, we evaluated the immunochromatographic assay NG-Test Carba 5v2 (NG-Biotech), with improved IMP variant detection on 31 IMP producers, representing the different branches of the IMP phylogeny, including 32 OXA-48, 19 KPC, 12 VIM, 14 NDM, and 13 multiple carbapenemase producers (CPs), 13 CPs that were not targeted, and 13 carbapenemase-negative isolates. All tested IMP variants were accurately detected without impairing detection of the other carbapenemases. Thus, NG-Test Carba 5v2 is now well adapted to countries with high IMP prevalence and to the epidemiology of CP-Pseudomonas aeruginosa, where IMPs are most frequently detected.

A new assay for global fibrinolysis capacity (GFC): Investigating a critical system regulating hemostasis and thrombosis and other extravascular functions.

For many years, the importance of fibrinolysis has been recognized, first for its intravascular antithrombotic action, and more recently for its many extravascular activities, associated with matrix degradation and tissue remodeling. In the blood circulation system, fibrinolysis prevents thrombosis, and is associated with various biological and clinical situations: risk factors for cardio-vascular diseases in high risk clinical situations (type II diabetes, hypertension, triglycerides, high BMI, elevated glucose, etc.), probably resulting from a significant reduction of the fibrinolysis potential, and elevation of PAI-1. Noteworthy, t-PA is mainly present as an inactive complex with PAI-1, and its concentration in plasma tends to follow that of PAI-1, but in a lesser extent. Hypofibrinolysis can favor the occurrence of thrombotic events, and possibly other biological dysfunctions. Fibrinolysis activity is however difficult to evaluate as it has a delayed activity after clot formation, is initiated and regulated after fibrin generation, and conversely to clotting, its action is delayed (long lag phase) and slow, before being dramatically amplified leading to rapid clot dissolution. We have designed a new assay for evaluating the global fibrinolytic capacity (GFC) in the body. Reagents are used in association with a specific instrument, which can be connected to any computer, and dedicated software is used for analyzing clot lysis kinetics. The assay is performed in a micro-cuvette, introduced into one of the instrument wells at 37 °C, and light transmittance is continuously measured. Assayed plasma is first supplemented with a limited and constant amount of t-PA with silica and is then clotted with thrombin and calcium. Clot dissolution (measurement of turbidity change) is recorded over time using the dedicated instrument (Lysis Timer), and clot lysis kinetics are analyzed with the associated software: primary and secondary derivatives of the light transmission curve give information on kinetics and completion of clot dissolution. Total assay time is about 1 h (but in the presence of hypofibrinolysis it can be prolonged). The concentration of t-PA used for the assay has been adjusted (100 ng/ml) to obtain an optimal sensitivity to hypofibrinolysis within a short time interval, and clot dissolution occurs within about 45 min for normal individuals, with a broad range from 30 min to 60 min, with some samples presenting a clot dissolution time >60 min (hypofibrinolysis). This new assay is performed with the tested plasma intrinsic factors, especially its own fibrinogen, and only exogeneous t-PA is added. GFC is highly sensitive to PAI-1 activity, but other factors regulating fibrinolysis contribute to the clot dissolution kinetics. Freshly prepared or frozen and thawed citrated plasma can be used. The usefulness of this assay for clinical applications is under investigation. Although fibrinolysis is mainly initiated in the body upon stimulation or blood clotting, and rapidly diluted and inhibited in the circulation, evaluation of its "residual" activity in plasma is expected to reflect its global body potential.

Update on functional and genetic laboratory assays for the detection of platelet microvesicles.

Functional and genetic assays for measuring platelet microvesicles (PMVs) are presented and discussed. Functional assays concern two groups of methods: a) homogeneous assays using the cofactor activity of phospholipids (PPLs) contained in PMVs and present in assayed plasmas, and a coagulation or a thrombin generation assay (TGA) as "end points"; b) capture-based assays, in which PMVs bind to an immobilized ligand, such as Annexin V in the presence of calcium, or monoclonal antibodies (MoAbs) specific for membrane proteins. Genetic assays aim to detect micro-RNA (miRNA) present in PMVs: miRNA must be extracted from plasma, and the expression pattern can be determined by various methods such as quantitative real-time PCR, microarray or sequencing. All these technical approaches introduce new exploration tools for measuring or quantitating PMVs or their associated activities, as biomarkers for disease evolution, their diagnosis or prognosis, and for monitoring of some antithrombotic or anti-inflammatory therapies. They offer invaluable analytical tools for research, drug discovery and epidemiological studies and have a strong potential as diagnostic tests.