Rapid Diagnostic of External Bacterial Endophthalmitis (EBE)

Abstract

Purpose: : EBE is most commonly noted following cataract or glaucoma filtering surgery, corneal transplantation and injections of steroids or inhibitors of vascular endothelial growth factor. Culture methods produce often negative results (21%-63%). The objective of this study was to improve the microbiological diagnosis of EBE and reduce the time needed to obtain results using a new real-time PCR (retiPCR).

Methods: : Samples were tested using classic methods (aero- anaerobic germ isolation and identification) and the new molecular retiPCR. The primers and TaqMan probes detect in one series of reactions >90% of the most representative EBE strains. For the other 10% the system allows to identify any kind of bacteria. DNA were isolated using the MagnaPure LC Isolation station (Roche). The method was designed to identify and semi quantify eubacterial DNA (all bacteria) and assess the load of Staphylococci, Streptococci, Acinetobacter, Stenomaltophilia, Pseudomonas, Enterobacteriacae, Propionibacterium, Corynebacterium and Taq-pol inhibitors. The hybridisation site of each probe was chosen avoiding overlapping and labelled and with dyes which absorption wavelengths differences of >20 nm(Operon,Germany). The PCR consisted in 1 cycle at 95°C for 20 sec and 45 at 95°C for 3 sec and 30 sec at 60°C. Each run contained negative controls and DNA extracts from the solutions used during the procedure.

Results: : For the different strains tested the detection capacity of the method was of at least 0.1 CFU/µl. In a series of 20 samples -EBE with clinical signs-, this reti PCR showed higher rates of positive (90%) than microscopic examination + culture (70%). The retiPCR procedure was conducted in a closed tube and the time to obtain results was reduced from 24-96 hours (or more for slow growth germs) to 2 hours.

Conclusions: : This new method allows detection and identification of most of bacteria responsible for EBE in one step with no need of any additional procedure (RFLP, DNA cloning and sequencing). In addition, for the characterization of strains, variants, etc. this new fast large spectrum real-time PCR produces amplicons that can be directly sequenced immediatly after.

Keywords: endophthalmitis • clinical laboratory testing • bacterial disease