A dot hybridization assay for the diagnosis of bacterial keratitis

PURPOSE: To evaluate a bacterial dot hybridization (BDH) assay for the diagnosis of bacterial keratitis (BK). METHODS: Sixty-one qualified corneal scrapings from 61 patients with suspected microbial keratitis were collected consecutively and prospectively. Among the 61 patients, 16 cases were BK and 45 cases were non-BK, including fungal keratitis, viral keratitis, parasitic keratitis, and non-microbial keratitis. Molecular diagnosis of BK in these corneal scrapes was performed using the BDH assay with three universal bacterial probes (PB1, PB2, and PB3) and three genus-specific probes (Aci, Klb, and Psu) to detect Acinetobacter, Klebsiella, and Pseudomonas, respectively. Signals were standardized after grayscale image transformation for objective validation using receiver operating characteristic (ROC) curves. RESULTS: The standardized intensities for the three universal probes differed statistically significantly between the BK group and the non-BK group. Based on the ROC curves, the sensitivities of PB1, PB2, and PB3 were 81.3%, 81.3%, and 93.8%, and the specificities were 71.1%, 88.9%, and 91.1%, respectively. The sensitivity and specificity of the Psu probe were 92% and 100%, respectively, while those of the Aci and Klb probes could not be estimated because there were no BK cases caused by Acinetobacter spp. or Klebsiella spp. CONCLUSIONS: The BDH assay is an effective molecular approach to improve the diagnosis of BK. Because the bias from bacterial contamination on the ocular surface can be minimized with signal standardization, the assay has the potential to be adopted for routine clinical practice.