IDENTIFICATION OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS IN SUBGINGIVALPLAQUE BY PCR

The purpose of this study was to assess the sensitivity and specificit y of the PCR in detecting Actinobacillus actinomycetemcomitans. The PC R's detection capability was compared with those of three other method s: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conve ntional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was am plified by PCR with primers TT-15 and TT-16. For CH, the PCR product w as labeled with digoxigenin and used as a hybridization probe, Nucleot ide sequence analysis of the PCR product of A. actinomycetemcomitans 1 D4 and 1664 and three clinical isolates revealed complete homology amo ng the tested strains, with only one base substitution (at position 13 44) in comparison with the published sequence. With artificially infec ted subgingival plaque, the detection limit of PCR for A. actinomycete mcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR ( CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of s ubgingival plaque samples from 35 patients with periodontal disease an d 10 periodontally healthy subjects revealed that CE-PCR and CH had th e highest overall rate of A., actinomycetemcomitans detection (both 58 %), followed by PCR and culture (both 42%). With CH as the ''gold stan dard,'' the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respective ly, The CE-PCR provided acceptable positive and negative predictive va lues (greater than or equal to 70%) when the prevalence of A., actinom ycetemcomitans varied between 30 and 70%, PCR alone provided comparabl e predictive values over a narrower range of prevalence rates (30 to 5 0%), while culture did not afford acceptable predictive values at any prevalence rate, PCR and CE-PCR were found to be superior to culture w ith presumptive biochemical identification and should be the preferred methods for the detection of A. actinomycetemcomitans in subgingival plaque.