After PCR amplification using universal primers for the 16S rDNA, a product of the appropriate size (1505 bp) was obtained from all samples examined, indicating that bacteria were present in all cases and demonstrating the suitability of the DNA for PCR and the absence of inhibitors in the reaction mixture.
In the 22 cases of asymptomatic periradicular lesions, T. denticola was detected in17 teeth (77.3%), T. socranskii in nine (40.9%), T. vincentii in four (18.2%) and T. pectinovorum in three (13.6%). Nineteen cases (86.4%) yielded at least one of the four species. In the cases of acute apical periodontitis showing spontaneous pain, T. denticola was found in five of six cases (83.3%) and T. socranskii in three of six (50%). None of these cases was positive for either T. vincentii or T. pectinovorum. In the total cases diagnosed as acute apical periodontitis,T. denticola was detected in eight cases (80%), whilst T. socranskii and T. vincentii were detected in four (40%) and one case (10%), respectively. No symptomatic case yielded T. pectinovorum. Eight cases were positive for at least one of the test species.
In general, species-specific nested PCR detected T. denticola in 25 of 32 cases (78.1%), T. socranskii in 13of 32 cases (40.6%),T. vincentii in five of 32 cases (15.6%) and T. pectinovorum in three of 32 cases (9.4%). At least one of the four Treponema species was found in 27 of the 32 cases examined (84.4%). Data regarding prevalence values are represented in Fig.1. The species T. denticola was associated with T. socranskii in 12 cases (eight asymptomatic and four symptomatic cases), with T. vincentii in four cases (three asymptomatic and one symptomatic cases) and with T. pectinovorum in two asymptomatic cases. T. socranskii was detected together with T. pectinovorum in two asymptomatic cases and with T. vincentii in two cases (one asymptomatic and another symptomatic).No case yielded a coinfection with T. pectinovorum and T. vincentii. Three cases yielded three of the treponemal species evaluated herein. The species T. denticola, T. socranskii and T. vincentii were concomitantly infecting two root canals (one asymptomatic and another symptomatic). Another asymptomatic case was positive for T. denticola, T. socranskii and T. pectinovorum.

Figure 1. Prevalence of four Treponema species in root canal infections. Data refer to the percentage of asymptomatic cases, symptomatic cases and the total number of cases positive for each target species. Data about Treponema refer to the cases where at least one of the four species was detected.