title: Development and evaluation of a saliva-based chair-side diagnostic for the detection of Porphyromonas gingivalis.
abstract: Porphyromonas gingivalis is a key pathogen in the polymicrobial biofilm that is associated with the oral disease chronic periodontitis. A number of studies have shown that in humans the level of P. gingivalis in the polymicrobial biofilm is positively correlated with disease progression. The aim of this study was to develop a P. gingivalis diagnostic that has high specificity and sensitivity for P. gingivalis using a range of laboratory and clinical isolates and then compare the efficacy of the diagnostic with RTPCR using samples from chronic periodontitis patients and age- and sex-matched healthy controls. Key parameters for the kit were to use saliva as the biological fluid as this is a most convenient medium for chair-side sampling and to give a positive reading for the reported threshold for detection of 5×10(5) P. gingivalis cells/mL that indicates disease progression. We initially screened a range of monoclonal antibodies for recognition of the P. gingivalis conserved virulence factor RgpA-Kgp complex and identified two mAbs that could be used in a capture and detection ELISA system. These mAbs were used to formulate and manufacture the GC P. gingivalis saliva diagnostic kit used in the study. To validate the saliva kit, saliva (P. gingivalis free) was spiked with known concentrations of viable P. gingivalis whole cells of W50, 381, A7A1-28, and ATCC 33277; P. gingivalis clinical isolates; P. gingivalis vesicles; and the secreted form of the RgpA-Kgp complex. Laboratory findings indicated that the kit was able to detect all laboratory and clinical isolate strains of P. gingivalis at 5×10(4)/mL to 5×10(5)/mL. It was also able to detect the RgpA-Kgp complex and vesicles at 5×10(4) and 5×10(5) cell equivalent doses, respectively. Saliva and plaque were then collected from 50 subjects with moderate-severe chronic periodontitis and 50 age- and sex-matched subjects with healthy periodontium. Real-time PCR was utilised to analyse levels of P. gingivalis in both saliva and plaque. The saliva kit was found to give a positive result within 90 seconds. Using point bi-serial correlation analysis, a significant (p=0.04) correlation was found for detection of P. gingivalis using the saliva kit and P. gingivalis levels in saliva and plaque as determined by real-time PCR. A sensitivity of 92% and a specificity of 96% were found when compared to real-time PCR at a 10(5) P. gingivalis cell threshold.In conclusion, the P. gingivalis saliva kit was shown to be rapid and has a comparable detection capacity to real-time PCR. Thus, the P. gingivalis saliva diagnostic has the potential to be a simple and time-efficient chair-side diagnostic for the detection of P. gingivalis.
authors.affiliation: Melbourne Dental School, Oral Health CRC, The University of Melbourne, Melbourne, Australia.
authors.forename: Neil M
authors.initials: NM
authors.lastname: O'Brien-Simpson
authors.affiliation: Melbourne Dental School, Oral Health CRC, The University of Melbourne, Melbourne, Australia.
authors.forename: Kate
authors.initials: K
authors.lastname: Burgess
authors.affiliation: Melbourne Dental School, Oral Health CRC, The University of Melbourne, Melbourne, Australia.
authors.forename: Gail C
authors.initials: GC
authors.lastname: Brammar
authors.affiliation: Melbourne Dental School, Oral Health CRC, The University of Melbourne, Melbourne, Australia.
authors.forename: Ivan B
authors.initials: IB
authors.lastname: Darby
authors.affiliation: Melbourne Dental School, Oral Health CRC, The University of Melbourne, Melbourne, Australia.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds