Background: Diagnosis of bacterial endophthalmitis (BE) often fails due to: 1-reduced volumes of vitreous fluid (VF) and aqueous humor (AH); 2-lack of sensitivity of culture; 3-antibiotic treatments; 4-PCR cross-contamination and 5-limitations for real-time PCR melting curve interpretation. We developed an fast real-time-PCR to improve laboratory diagnosis performances.
Methods: a-PBS, VF, AH and cell suspensions spiked with Bacteria; b-VF and AH from endophthalmitis, and c-VF and AH from non-infective disorders were processed after adding an internal control (IC). DNA was extracted (MagnaPure®) and introduced into 4 tubes containing selected primers and probes for the identification and quantification of all Bacteria (Bac) and of 8 Genera by fast real-t PCR (f-real-t PCR). Performances of diagnosis based on direct microscopic examination, culture and f-real-t PCR were compared.
Results: The f-real-t PCR detected at least 0.01 CFU Bac/µl with no cross reactivity with fungi. Correlation with culture positive results was 100%. Sixty % of BE samples tested culture positive but f-real-t PCR tested positive for 90%. Samples from non-infective cases were negative.
Conclusion: The f-real-t PCR detects and quantifies Bac, Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter, Propionibacteriacae and Corynebacteria in one run. Cultures required several hours to days (with non-negligible number of false negative results) and the f-real-t PCR was completed in 90 minutes. The f-real-t PCR appears as a new tool for the diagnosis of BE and its usefulness requires validation with larger series of samples.