Date Published: March 11, 2019
Publisher: Public Library of Science
Author(s): Sarah Benabdelkader, Julien Andreani, Alexis Gillet, Elodie Terrer, Marion Pignoly, Herve Chaudet, Gerard Aboudharam, Bernard La Scola, Peter Eickholz.
Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases.
Periodontal disease is a widespread oral disease affecting adults and younger people, characterized by an inflammatory reaction that affects periodontium tissue . A new classification published in 2018 based on description (localized or generalized), severity and complexity of management divides periodontitis into 4 stages, including initial periodontitis (I), moderate periodontitis (II), severe periodontitis with potential for additional tooth loss (III) and advanced periodontitis with extensive tooth loss and potential for loss of dentition (IV) . According to this classification, periodontitis is also graded in 3 levels estimated with direct or indirect evidence of progression rate: slow (A), moderate (B) and rapid (C). Periodontal disease is characterized by receding gums, alveolar bone destruction, loss of dental junctions associated with the apparition of periodontal pockets, and in some forms, dental calculus deposits. This promotes the establishment of an anaerobic microenvironment that allows the growth of anaerobic microorganisms . The immunological process initiates the migration of microorganisms into tissues and disrupts the immune response, causing the periodontium to resorb [4,5]. Some host risk factors have now been clearly identified, including smoking  and diabetes mellitus , but other genetic factors require further study .
In this study, by combining a polyphasic approach that associates culture and qPCR, we found a correlation between periodontitis and the presence of T. tenax (p< 0.05). Although T. tenax is more frequently detected by qPCR in patients than in controls, the difference is not significant (p = 0.435). A significant difference was observed using culture only (p = 0.015). By combining the culture and PCR results to neutralize the effect of false negative of each technique and evaluate the real prevalence of T. tenax, the difference is significant (p = 0.042). The probability of false positive/negative frequency is reduced due to the good correlation observed between both techniques: 83% of positive cultures also positive for qPCR and 88% of negative cultures also negative for q-PCR techniques. We believe that this good correlation indicates that the culture and handling protocols, including the transport medium specifically developed for anaerobic microorganisms, were highly efficient. However, as commonly observed in clinical microbiology, the higher sensitivity of the PCR suggests that some T. tenax did not grew in culture. The reasons are unknown but usually because microorganisms are dead at time of inoculation due to delayed inoculation between sampling and culture, quality of the operator or quality of the batch of transport or culture media. The 11.6% of the positive samples in culture not identified by real-time PCR shows that false positive occur also with molecular amplification, usually as a consequence of inhibitors. A high prevalence of T. tenax in both controls and patients is detected using genomic-dependent and culture-based methods of detection. T. tenax was more frequently associated with severe periodontitis. Three clusters of strains were highlighted by the MLST genotyping system, two were significantly associated with periodontitis. T. tenax appears to be associated with the onset or/and evolution of periodontal diseases. However, although these differences are statistically significant, it is impossible to determine whether they are a cause or a consequence of the disease. Source: http://doi.org/10.1371/journal.pone.0213338