Diagnostic accuracy of multiplex real-time PCR approaches compared with cultivation -based detection methods: Monitoring the endopathogenic microbiota pre and post photo-activated disinfection.

Abstract

Several microbial species have been implicated in the pathogenesis of endodontic diseases that colonize the infected root canal system. Since the complete removal of endopathogenic agents is essential in endodontic infection therapy, photo-activated disinfection (PAD) is suggested as an alternative method to traditional antimicrobial therapy. Recent studies reported that the molecular methods with low sensitivity and high efficiency to identify fastidious anaerobic endopathogenic microbiota can be replaced by the cultivation-based approaches. This study aimed to validate the multiplex real-time PCR in order to identify six common microorganisms associated with the endodontic infections before and after the PAD. Microbial specimens from the root canals of 50 patients with primary and secondary endodontic infections were collected before PAD treatment using sterile paper points. Toluidine blue O (TBO)-mediated PAD was performed on the root canals, followed by resampling. The prePAD- and postPAD-treatment endodontic samples were transferred to a transport medium and six target microorganisms were then identified from the samples using the microbiological culture techniques and multiplex real-time PCR approach. Multiplex real-time PCR could represent the presence of all target microorganisms in 100% cases before and after the PAD. Before PAD, using the culture method, Enterococcus faecalis (100%) was found to be the most frequent, followed by Veillonella parvula (97.5%), Aggregatibacter actinomycetemcomitans (94.7%), Porphyromonas gingivalis (84.3%), Lactobacillus rhamnosus (84.3%), and Actinomyces naeslundii (66.6%); whereas, after PAD these microbial frequencies changed to 80%, 83.3%, 66.6%, 80%, 66.6%, and 33.3%, respectively. The sensitivity and negative predictive value of the multiplex real-time PCR were 100% before and after the PAD, whereas the highest and the lowest specificities were 100% and 82% before PAD, and 97% and 89% after PAD for E. faecalis and P. gingivalis, respectively. The highest (100%) and the lowest (66%) positive predictive values were for V. parvula and A. naeslundii before and after the PAD, respectively. As observed from the results, multiplex real-time PCR demonstrated high sensitivity and specificity when compared to the culture technique. Therefore, it can prove to be a highly sensitive technique to detect the endodontic infections microflora.