Detection and Gram Discrimination of Bacteria from Keratitis by Real-Time PCR Assay

Abstract

Purpose : The purpose of this study was to validate a multiplex real-time PCR assay for detection of bacteria directly from corneal samples collected from patients with clinical diagnosis infectious of keratitis and compare with the smears results stained by Gram and cultures

Methods : A total of 70 corneal samples were collected from 67 patients and were included in this study. Smears for Gram stain and cultures on Solid Agar plates: Sabouraud, Blood and Chocolate. The DNA extraction was performed by QIAamp DNA mini kit (Qiagen, Hilden, Germany). Analytical sensitivity of Duplex Gram-Specific TaqMan-Based PCR was determined by a 10-fold dilution of 0.5 McFarland suspension of Staphylococcus aureus and Escherichia coli.

Results : The limit of detection of S. aureus and E. coli was 10² and 10³ dilution, respectively. From the 70 samples included in the study 38 were positive to bacteria by culture (27 Gram positive and 11 Gram negative), only 8 of these samples were positive on smears. Real-time PCR was positive on 21 of the 38 samples culture positive for bacteria. Real-time PCR was positive on 18 of the 30 samples that were negative on Gram

Conclusions : Real-time PCR was more sensitive than Gram for bacterial detection. When compared to culture results, the real-time PCR was less sensitive. These results where different than expected, probably because of the presence of PCR inhibitors in the samples collected directly from the cornea (anesthetic used for sample collection) and the sample collection for PCR was the last one to be performed, after smears and cultures.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.