Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain D-amino acids, led to the discovery of five P. gingivalisspecific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and L-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10 5 CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of L-amino acids and Bz-L-Arg-NHPhNO 2 showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single D-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of D-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Galassi F, Kaman WE, Anssari Moin D, van der Horst J, Wismeijer D, Crielaard W, Laine ML, Veerman ECI, Bikker FJ, Loos BG. Comparing culture, real‐time PCR and fluorescence resonance energy transfer technology for detection of Porphyromonas gingivalis in patients with or without peri‐implant infections. J Periodont Res 2012; 47: 616–625. © 2012 John Wiley & Sons A/S Background and Objective:  The aim of the study was to compare the detection of Porphyromonas gingivalis using a fluorescence resonance energy transfer (FRET) technology with commonly used diagnostic methods in salivary and subgingival plaque samples from subjects with dental implants. P. gingivalis was considered as a marker for a pathogenic microbiota. Material and Methods:  Ninety‐seven adult subjects were recruited, including periodontally healthy controls with no dental implants, implant controls with no peri‐implant disease and patients with peri‐implant disease. Saliva and subgingival/submucosal plaque samples were collected from all subjects and were analyzed using culture, real‐time PCR and FRET technology employing P. gingivalis‐specific substrates. Results:  It was found that the P. gingivalis‐specific substrates were highly suitable for detecting the presence of P. gingivalis in saliva and in subgingival plaque samples, showing comparable specificity to culture and real‐time PCR. Conclusion:  We applied the FRET technology to detect P. gingivalis in implant patients with or without an implant condition and in controls without implants. The technique seems suitable for detection of P. gingivalis in both plaque and saliva samples. However, with all three techniques, P. gingivalis was not very specific for peri‐implantitis cases. Future work includes fine‐tuning the FRET technology and also includes the development of a chair‐side application.