Validation of a multiplex qPCR assay for detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque samples. A comparison with anaerobic culture

Abstract

ObjectiveTo validate a multiplex real time qPCR (m-qPCR) assay for the simultaneous detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival samples, when compared with anaerobic culture. Material and methodsSubgingival plaque samples were obtained from patients seeking periodontal treatment. Samples were processed in parallel by anaerobic culturing and by m-qPCR directed to the target bacterial species. Counts and frequency of detection were calculated and analyzed by Mann-Whitney U and chi-square tests, respectively. Contingency tables were constructed, and sensitivity, specificity, predictive values and Lin’s correlation coefficients were calculated. ResultsFifty-nine samples were included in the study. A good concordance was achieved between m-qPCR and culture for A. actinomycetemcomitans and P. gingivalis (net agreement, 94.92% and 91.53%, respectively). For T. forsythia, m-qPCR showed statistically significant higher counts than culture (p < 0.005), and low specificity (3.12%) and concordance (47.46%). High sensitivity (above 96.22%) was attained for the three target bacteria with m-qPCR. ConclusionCompared to culture, the tested m-qPCR assay for subgingival plaque samples showed high degree of sensitivity in the simultaneous quantification of A. actinomycetemcomitans, P. gingivalis and T. forsythia.