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  • Title: Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays.
    Author: Bispo PJ, de Melo GB, Hofling-Lima AL, Pignatari AC.
    Journal: Invest Ophthalmol Vis Sci; 2011 Feb 22; 52(2):873-81. PubMed ID: 20702819.
    Abstract:
    PURPOSE: To develop and apply real-time PCR protocols to the detection and classification of the Gram status of bacterial pathogens in aqueous and vitreous humor collected from clinically suspected intraocular infections. METHODS: The analytical specificity of two PCR assays, SYBR Green 16S rDNA-Based Universal PCR (SGRU-PCR), and a Multiplex Gram-Specific TaqMan-Based PCR (MGST-PCR), was determined with 31 clinically important pathogens, including 20 Gram-positive and 11 Gram-negative. Analytical sensitivity was determined with a 10-fold dilution of Staphylococcus epidermidis and Escherichia coli DNA. Assays were further tested on aqueous (n = 10) and vitreous humor (n = 11) samples collected from patients with clinically diagnosed intraocular infections. RESULTS: DNA was amplified from all control bacterial isolates when using SGRU-PCR. MGST-PCR correctly classified the Gram status of all these isolates. The SGRU-PCR limit of detection of S. epidermidis and E. coli DNA was 100 fg/μL (E = 0.82 and 0.86; r(2) = 0.99) and for MGST-PCR, 1 pg/μL (E = 0.66 and 0.70; r(2) = 0.99. For clinical intraocular samples, positivity of culture was 47.6% and for real-time PCR assays, 95.2%. Gram classification was achieved in 100% of MGST-PCR-positive samples. Among microbiologically negative samples, real-time PCR assays were positive in 90% of cases. The false-positive rate in control aqueous was 3.2%, and control samples of vitreous were negative. CONCLUSIONS: The real-time PCR assays demonstrated good correlation, with culture-proven RESULTS: With the use of these methods, bacterial detection was improved from 47.6% to 95.3%, demonstrating them to be sensitive, rapid tests for diagnosis of bacterial endophthalmitis.
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