Effectiveness of 16S ribosomal DNA real-time PCR and sequencing for diagnosing bacterial keratitis

Purpose To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis. Methods We studied 272 eyes of 272 patients with keratitis. Eyes with keratitis were classified as “definite” ( N = 118), “likely” ( N = 71), or “non-bacterial” ( N = 83) to have bacterial keratitis. The diagnostic efficacy of real-time PCR and conventional testing was determined by receiver operating characteristic analysis. The copy numbers of bacterial DNA and clinical characteristics were retrospectively analyzed for association with concordant culture results in the “definite” cases. Results The level of bacterial DNA was significantly associated with the diagnostic probability of the three diagnostic categories. The level of bacterial DNA had comparable diagnostic efficacy with the area under the curve (AUC) at 0.67, by culture at 0.65, and by smear testing at 0.73. The efficacy was significantly improved by combining the DNA level with the conventional culture testing with an AUC of 0.81. Analysis of the “definite” cases showed culture positivity in 51.8% (58 eyes), and of these, 41 eyes (70.7%) were higher than the cutoff PCR values and 40 eyes were identified by 16S r-DNA sequencing. In the culture-negative eyes, the level of bacterial DNA was significantly lower ( P = 0.0008). Eyes with higher bacterial DNA levels had significantly concordant outcomes with sequencing and culture results ( P = 0.006). Previous antibiotic treatments decreased the bacterial DNA amount by 0.09-fold, and it was a significant factor for discordance ( P = 0.006). Conclusion Quantification of the bacterial DNA level and conventional testing improves the diagnostic efficacy of infectious bacterial keratitis.