Sampling of periodontal pathogens by paper points: evaluation of basic parameters

Paper points are widely established for the collection of subgingival plaque or other oral samples to analyze the microbiota, especially the presence of periodontal pathogenic bacteria such Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis. In contrast to the high frequency of usage of paper points in oral sampling, very few data are available about the parameters influencing the sampling process. Therefore, in four different in vitro experiments (6-9 repeats), we inoculated paper points with standardized suspensions (2 x 10(9) colony-forming units/ml) of A. actinomycetemcomitans and P. gingivalis to test the influence of the origin (kind) of paper point (''manufacturer"), size (according to the International Organization for Standardization ISO 25-80), sampling time (5 to 60 s), and elution time (5 to 60 s). Sampled bacteria were detected and (semi-) quantified using 16S rRNA/DNA-directed oligonucleotide probes. The bacterial load was categorized and calculations performed with index values ranging between 0 (below the detection limit of 10(2) to 10(3) colony-forming units) and 9 (>10(6) colony-forming units). We found statistically significant differences in the efficiency of bacterial sampling between the 5 paper point manufacturers tested, expressed in a mean bacterial index between 4.4 and 7.8. ISO 45 Paper points were statistically proven to work most efficiently. According to our results, a sampling time of 60 s seems to be optimum. However, shorter times between 5 and 30 s did not significantly reduce the sampling efficiency. We found an interval of 20 s best to elute bacteria from the paper points by vortexing. The evaluation of basic parameters for subgingival plaque sampling by paper points might help to optimize microbiologically based diagnostics in periodontal diseases.