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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.

Purpose To develop a PCR gel analysis method for assessing the bacterial bioburden in orthokeratology contact lens (OK) case fluid determined by culture. Methods A prospective study with the participation of 41 OK wearers (20 girls, 21 boys) was performed. The mean OK-wearing experience was 3.5±1.9 years. PCR was used to assess the bacterial bioburden (colony-forming units per milliliter) of OK after removal and soaking in the storage case for 6 h. The signal intensity of the PCR bands was analyzed after grayscale image transformation. The difference (cPCR-d) and ratio (cPCR-r) between a PCR signal and its background were used as two standardized indices of PCR signals. The association between the two indices of the PCR signals and the bacterial bioburden determined by culture were analyzed with Pearson’s correlation coefficient (r) and receiver operating characteristic (ROC) plots. Results At least one microbe was isolated from the OK lens case from 38 of the 41 subjects. Both cPCR-d and cPCR-r showed strong correlations with the bacterial bioburden (r>0.7, p