Molecular detection and genotyping of Porphyromonas Gingivalis directly from oral rinse: Implications for Periodontitis pathogenesis

Abstract

Porphyromonas gingivalis is a Gram-negative anaerobe implicated as a primary etiological agent in chronic periodontitis, characterized by progressive inflammation and degradation of periodontal structures. The bacterium's pathogenicity is closely associated with its expression of distinct fimbrial subunits encoded by the fimA gene, which serves as a pivotal marker for strain differentiation and genotyping. Profiling the distribution of fimA genotypes in clinical specimens is vital for elucidating the pathogen's role in the etiology and progression of periodontitis. In this study, we developed a highly sensitive and specific real-time PCR assay utilizing TaqMan probe technology for the direct detection and genotyping of P. gingivalis fimA variants in oral rinse samples. The assay was designed to investigate both the prevalence and distribution of the six fimA genotypes (I, Ib, II, III, IV, and V) among individuals with periodontitis compared to periodontally healthy controls. A cohort of 30 participants, comprising 12 healthy individuals and 18 patients diagnosed with periodontitis, provided oral rinse samples for analysis. The real-time PCR assay, employing genotype-specific primers and TaqMan probes, demonstrated robust performance in discriminating the six recognized fimA genotypes. The assay achieved a detection rate of 94.4% in periodontitis samples, accurately identifying all fimA genotypes directly from the oral rinse specimens. The most prevalent fimA genotype identified was Ib, occurring in 33.33% of the samples. This was followed by genotype II, with a frequency of 27.78%. Genotype IV was found in 16.67% of samples, and genotype I was present in 5.56% of samples. Additionally, 16.67% of the samples contained combinations of two fimA types, including II,Ib; II,III; and II,IV. Among these, genotype II was the most frequently detected in combination with other genotypes. The linear regression analysis yielded a slope of -3.42, corresponding to an amplification efficiency of 94.5%, underscoring the assay's precision and reliability. The data indicate a potential contributory role of fimA genotypes Ib and II in periodontitis pathogenesis, establishing the developed PCR assay as a valuable diagnostic and epidemiological tool for monitoring P. gingivalis in clinical settings.