The diode laser fluorescence device was introduced to diagnose caries lesions in occlusal and smooth surfaces. Despite promising results, much variation has been found in the protocol of utilization. The goal of this study was to evaluate the influence of dental plaque and drying time on the laser fluorescence (DIAGNOdent) performance in occlusal sites of primary molars. For the drying time study, 63 suspected occlusal sites in primary molars were used. The same sites were evaluated in four different conditions: (1) moist teeth, (2) teeth dried for 3 s with a 3-in-1 syringe, (3) teeth dried for 15 s, and (4) dehydrated teeth. For the plaque experiment, we selected 28 occlusal sites. After the baseline laser fluorescence readings, plaque collected from children was placed on the suspected site. New laser fluorescence measurements were performed. For both studies, we compared the laser fluorescence values, as well as sensitivity, specificity, accuracy, and area under ROC curve. For different drying times, the more dehydrated the teeth, the higher the values. Nevertheless, the performance of the method did not change significantly, except for dehydrated teeth. In the presence of plaque, the laser fluorescence values were reduced, and the evaluated parameters were worse than with no plaque. In conclusion, the hydration state of the teeth did not change the results significantly, but the presence of plaque worsened the performance of the laser fluorescence method.

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