This study demonstrates the usefulness of Q-PCR for enumerating putative pathogens in clinical periodontal specimens and that the numbers of the three organisms in all sites decrease with non-surgical periodontal therapy.

Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the signi®cance of their relative numbers and progress of the disease is still unclear. Traditional quantitative methods are generally time-consuming and inaccurate. The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P. gingivalis, Pr. intermedia and A. actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes were employed: the ®mbrial gene ( ®mA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitans. Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template. Coampli®cation of target and competitive templates yielded products of expected size and non-speci®c recognition by the primers was not found. The sensitivity of the designed primers was 100 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. actinomycetemcomitans. The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r). The ®ndings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.

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Evidence that the cytolethal distending toxin locus was once part of a genomic island in the periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans strain Y4 The authors have previously shown that the periodontal pathogen Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 contains an operon for a genotoxin known as the cytolethal distending toxin (Cdt). The cdt locus in strain Y4 is flanked by remnants of heterologous plasmid and integrase sequences. In this study, the DNA sequence immediately downstream from the cdt locus on the Y4 chromosome was examined. The extended sequence contained a region that had all the characteristics of a typical bacterial pathogenicity or genomic island. The genomic island (GIY4-1) was approximately 22 kb long, was flanked by a bacteriophage attachment (att) sequence and contained a full-length integrase/resolvase gene (xerD). A total of 22 complete and partial ORFs represented putative DNA replication/DNA binding/conjugation proteins as well as hypothetical proteins. GIY4-1 was most closely related to putative genomic islands in Haemophilus ducreyi 35000HP and Haemophilus influenzae 86-028NP and to a chromosomal region in Haemophilus somnus 129PT. GIY4-1 was not present in HK1651, which was used as the prototype strain for genomic sequencing of A. actinomycetemcomitans. Several sequences in GIY4-1 were homologous to ORFs found on the A. actinomycetemcomitans plasmid pVT745. None of the identified ORFs in GIY4-1 appeared to encode potential virulence genes. However, several unique observations supported the possibility that the cdt locus of A. actinomycetemcomitans Y4 was originally contained within the genomic island. INTRODUCTIONActinobacillus actinomycetemcomitans is a facultative Gramnegative bacterium that has long been associated with localized aggressive periodontitis (LAP) and may also contribute to chronic forms of the disease. This bacterium has recently been reclassified as Aggregatibacter actinomycetemcomitans (Norskov-Lauritsen & Kilian, 2006). The bacterium expresses a number of genes whose products can be defined as virulence factors based on their in vitro biological activities or similarities to proteins produced by other pathogens (Henderson et al., 2003). Most notable is the expression of two multi-gene cytotoxin loci, leukotoxin (lkt) and cytolethal distending toxin (cdt). While Lkt has been extensively studied (see Lally et al., 1999 for a review), Cdt is a relatively newly described A. actinomycetemcomitans virulence factor. Cdt is a heterotrimer composed of three gene products (CdtA, CdtB and CdtC) that have homologues in a handful of facultative or microaerophilic Gram-negative pathogenic bacterial genera (Mao & DiRienzo, 2002;Mayer et al., 1999;Sugai et al., 1998).Although Cdt is not unique to A. actinomycetemcomitans, this bacterium is the only member of the oral microbial flora identified to date that carries and expresses the toxin locus (Yamano et al., 2003). Cdt is prevalent in A. actinomycetemcomitans strains. Fort...

Enumeration of specific periodontopathogens in subgingival plaque samples has been problematic because of either lack of sensitivity, specificity or the time taken to identify the organisms. These problems can be overcome using PCR, but quantification by this technique is more difficult. We report a simple quantitative PCR method developed for enumerating Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in clinical samples. The method relies upon inclusion of ethidium bromide in a closed-tube PCR reaction and measurement of resultant amplicons by quantifying the fluorescence generated when UV-light is passed through the walls of the amplification tube. Hence, both PCR and detection are performed in the same tube. The technique was compared with a quantitative competitive PCR and a commercial colorimetric quantitative PCR and proved to be at least as sensitive, specific and reliable for enumeration of the target bacteria. However, its speed and convenience make it particularly useful for large-scale analyses in both clinical laboratories and epidemiological studies.

Human papillomaviruses (HPVs) remain a serious world health problem due to their association with cervical and head and neck cancers. While over 100 HPV types have been identified, only a few subtypes are associated with malignancies. HPV 16 and 18 are the most prevalent oncogenic types in head and neck cancers. Although it has been proven that some subsets of benign and malignant head and neck lesions are associated with HPV, the general population have very little awareness and knowledge of their association with HPV. Therefore, the purpose of this study was to determine the knowledge of HPV and its links with head and neck benign and malignant lesions in a group of Pakistani dental patients who attended the Dental Department of the Sandeman provincial hospital in Quetta, Pakistan. One hundred and ninety-two patients were recruited and requested to answer a questionnaire. It was revealed that there was a low level of knowledge about HPV and its association with head and neck benign and malignant lesions among the participants. This result suggested that more education regarding the relationship of HPV in inducing head and neck benign and malignant lesions is required in this group of patients.