References (Microbial Signatures of Periodontal Disease: Quantifying the Key Pathogens)

Marin, M. J., Figuero, E., Herrera, D. & Sanz, M. Quantitative analysis of periodontal pathogens using real-time polymerase chain reaction (PCR). Methods Mol. Biol. 1537, 10.1007/978-1-4939-6685-1_11 (2017).
qPCR enables sensitive detection and quantification of periodontal pathogens including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, Fusobacterium spp. The method allows multiplex detection, provides proportional fluorescence output, and includes strategies to overcome common technical challenges.

Takahashi, K., Cunha, R. F. & Jardim, E. G. Periodontal pathogen colonization in young children by PCR quantification – a longitudinal survey. J. Clin. Pediatr. Dent. 41, 10.17796/1053-4628-41.6.7 (2017).
Longitudinal PCR analysis in children (6–24 months) revealed early colonization by periodontal pathogens correlated with maternal microbiota. Preventive biofilm control from infancy was suggested to reduce acquisition of aggressive bacteria.

Coffey, J., Choudhry, M., Shlossman, M., Makin, I. R. S. & Singh, V. K. Multiplex real-time PCR detection and relative quantification of periodontal pathogens. Clin. Exp. Dent. Res. 2, 10.1002/cre2.37 (2016).
Multiplex PCR was developed for simultaneous detection of A. actinomycetemcomitans, F. nucleatum, P. gingivalis, T. denticola, T. forsythia, demonstrating high specificity and proportional amplification, making it a practical tool for clinical bacterial profiling in periodontitis.

Marin, M. J., Ambrosio, N., Virto, L., Diz, P., Álvarez, M., Herrera, D., Sanz, M. & Figuero, E. Detection and quantification of A. actinomycetemcomitans, P. gingivalis and S. oralis in blood samples with different microbiological identification methods: An in vitro study. Arch. Oral Biol. 74, 10.1016/j.archoralbio.2016.11.007 (2017). Direct anaerobic culture (DAC) outperformed BACTEC, LC, and qPCR for detecting target pathogens in blood. The study highlighted methodological differences in sensitivity and specificity among culture and molecular approaches.

Gaertig, C., Niemann, K., Berthold, J., et al. Development of a point-of-care device for fast detection of periodontal pathogens. BMC Oral Health 15, 10.1186/s12903-015-0155-y (2015).
A novel point-of-care PCR device was developed for rapid detection of periodontal pathogens. Low sample volume, lyophilized reagents, and rapid analysis allow early diagnosis and personalized periodontal therapy.

Herrera, B. S., Henz, S. L., Dua, S., et al. Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT). Arch. Oral Biol. 152, 10.1016/j.archoralbio.2023.105721 (2023).
The ROQT approach identified both known pathogens such as T. forsythia and uncultivated species like F. alocis and Desulfobulbus sp. HMT 041. Severe periodontitis sites showed the highest pathogen loads, demonstrating the method’s ability to detect previously unrecognized oral bacteria.

Lages, E. J. P., Costa, F. O., Cortelli, S. C., et al. Alcohol consumption and periodontitis: Quantification of periodontal pathogens and cytokines. J. Periodontol. 86, 10.1902/jop.2015.150087 (2015).
Alcohol dependence was associated with higher subgingival levels of P. intermedia, E. corrodens, F. nucleatum, and proinflammatory cytokines (IL-1β). These results suggest negative effects of alcohol on both microbiologic and immunologic periodontal parameters.

Yoshida, R. A., Lobato, T. B., Gorjão, R., et al. Detection and quantification of pathogens in saliva of adolescents with cerebral palsy: a cross-sectional study. Front. Dent. Med. 4, 10.3389/fdmed.2023.1208243 (2023).
Adolescents with cerebral palsy showed variable detection of P. gingivalis and significant differences for P. intermedia, highlighting heterogeneity in pathogen colonization and potential periodontal risk.

Imamura, K., Takayama, S., Saito, A., et al. Evaluation of a novel immunochromatographic device for rapid and accurate clinical detection of P. gingivalis in subgingival plaque. J. Microbiol. Methods 117, 10.1016/j.mimet.2015.07.002 (2015).
The device demonstrated high sensitivity and specificity for rapid, semi-quantitative detection of P. gingivalis, correlating strongly with PCR-based measurements and allowing chair-side application.

Atieh, M. A. Accuracy of real-time polymerase chain reaction versus anaerobic culture in detection of A. actinomycetemcomitans and P. gingivalis: A meta-analysis. J. Periodontol. 79, 10.1902/jop.2008.070668 (2008).
Meta-analysis confirmed real-time PCR has high diagnostic accuracy for detecting key periodontal pathogens, supporting its use as a reliable alternative to culture.

Fujise, O., Miura, M., Hamachi, T. & Maeda, K. Risk of P. gingivalis recolonization during early periodontal maintenance in initially severe periodontitis sites. J. Periodontol. 77, 10.1902/jop.2006.050225 (2006).
Severe periodontitis sites before treatment were more prone to early recolonization by P. gingivalis, emphasizing the importance of close monitoring during maintenance therapy to prevent recurrence.

Alagl, A. S. Microbial pathogens associated with proximal dental caries in the primary dentition and their association with periodontal disease in children. Saudi J. Med. Med. Sci. 4, 10.4103/1658-631x.178323 (2016).
High bacterial loads of Fusobacterium nucleatum (Fn), Capnocytophaga (Ca), Campylobacter rectus (Cr) were associated with proximal caries and periodontal disease in children. DNA microbial probe testing allowed accurate identification and quantification, highlighting its utility in pediatric microbial diagnostics.

Marin, M. J., Figuero, E., Herrera, D., Sanz, M. Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR). Methods Mol. Biol. 2588, 10.1007/978-1-0716-2780-8_10 (2023).
qPCR enables sensitive quantification of P. gingivalis, A. actinomycetemcomitans, T. forsythia, C. rectus, S. oralis, Fusobacterium spp. Multiplex protocols and troubleshooting guidance make this a robust tool for clinical and research applications.

Vlasa, A., Biriș, C., Lazăr, L., et al. Detection and Quantification of Periodontopathogenic Bacteria in Subgingival Plaque Samples on Patients Undergoing Orthodontic Treatment. J. Interdiscip. Med. 1, 10.1515/jim-2016-0034 (2016).
PCR and DNA hybridization identified prevalent pathogens including A. actinomycetemcomitans, P. gingivalis, P. intermedia, Bacteroides forsythus, Treponema denticola, with highest loads correlating to disease progression in orthodontic patients.

Bedran, T. B. L., de Oliveira, G. J. P. L., Spolidorio, L. C., et al. Comparison of two different methods for detecting periodontal pathogenic bacteria. Braz. J. Oral Sci. 15, 10.20396/bjos.v15i3.8649599 (2016).
qPCR showed higher sensitivity than conventional PCR for detecting low bacterial loads of P. gingivalis, T. forsythia, P. endodontalis, supporting its use for quantification and monitoring post-therapy changes.

Su, Y., Huang, S., Hong, L., et al. Establishment of the molecular beacon-loop-mediated isothermal amplification method for the rapid detection of P. gingivalis. J. Microbiol. Methods 160, 10.1016/j.mimet.2019.01.013 (2019).
MB-LAMP provided rapid, highly sensitive, and specific detection of P. gingivalis at the point-of-care, with performance comparable to real-time qPCR but significantly faster, enabling early clinical diagnosis.

Gaertig, C., Niemann, K., Berthold, J., et al. Development of a point-of-care-device for fast detection of periodontal pathogens. BMC Oral Health 15, 10.1186/s12903-015-0155-y (2015).
A PCR-based point-of-care device was developed for rapid identification of periodontal pathogens, requiring low sample volumes and allowing personalized therapy by assessing microbial composition and load.

Boutaga, K., Van Winkelhoff, A. J., Vandenbroucke-Grauls, C. M. J. E., Savelkoul, P. H. M. Comparison of Real-Time PCR and Culture for Detection of P. gingivalis in Subgingival Plaque Samples. J. Clin. Microbiol. 41, 10.1128/JCM.41.11.4950-4954.2003 (2003).
Real-time PCR detected P. gingivalis in 77.8% of samples vs 56.3% by culture, demonstrating superior sensitivity and reliability of molecular techniques over conventional culture methods.

Udoh, S., Adukwu, E., Varadi, A., Saad, S. Effectiveness of the Human Oral Microbe Identification Microarray in Identifying Periodontal Pathogens: A Systematic Review. Appl. Microbiol. 2(3), 10.3390/applmicrobiol2030047 (2022).
HOMIM microarray provided robust, high-sensitivity identification and quantification of periodontal pathogens, outperforming earlier molecular and culture-based techniques and enabling detection of low-abundance and novel species.

Boutaga, K., Van Winkelhoff, A. J., Vandenbroucke-Grauls, C. M. J. E., Savelkoul, P. H. M. The additional value of real-time PCR in the quantitative detection of periodontal pathogens. J. Clin. Periodontol. 33(6), 10.1111/j.1600-051X.2006.00925.x (2006).
RT-PCR provided rapid, sensitive detection and quantification of key pathogens including P. gingivalis, T. forsythia, P. intermedia, Fusobacterium spp., capturing low-abundance species undetectable by culture.

Ishii, Y., Imamura, K., Kikuchi, Y., et al. Point-of-care detection of T. forsythia using an antigen-antibody assisted dielectrophoretic impedance measurement method. Microb. Pathog. 82, 10.1016/j.micpath.2015.03.017 (2015).

AA-DEPIM allowed rapid, chair-side detection of T. forsythia in saliva with high sensitivity (76.5%) and specificity (100%), correlating with PCR-based methods.

Doungudomdacha, S., Rawlinson, A., Walsh, T. F., Douglas, C. W. I. Effect of non-surgical periodontal treatment on clinical parameters and the numbers of P. gingivalis, P. intermedia and A. actinomycetemcomitans. J. Clin. Periodontol. 28(5), 10.1034/j.1600-051x.2001.028005437.x (2001).
Q-PCR quantified reductions (86–99%) in pathogen levels post non-surgical therapy, confirming treatment efficacy while highlighting persistent colonization of some species.

Sánchez, M. C., Marín, M. J., Figuero, E., et al. Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens. J. Periodontol. Res. 49(1), 10.1111/jre.12073 (2014).
PMA-qPCR selectively quantified viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum after antimicrobial exposure, demonstrating its potential for evaluating treatment effects in biofilms.

Galarraga-Vinueza, M. E., Dohle, E., Ramanauskaite, A., et al. Anti-inflammatory and macrophage polarization effects of Cranberry Proanthocyanidins (PACs) for periodontal and peri-implant disease therapy. J. Periodontol. Res. 55(6), 10.1111/jre.12773 (2020).
Cranberry PACs reduced pro-inflammatory cytokines (IL-6, IL-8) and promoted M2 macrophage polarization, suggesting anti-inflammatory benefits in periodontal therapy.

Ata-Ali, J., Flichy-Fernández, A. J., Alegre-Domingo, T., et al. Impact of heavy smoking on the clinical, microbiological and immunological parameters of patients with dental implants: a prospective cross-sectional study. J. Investig. Clin. Dent. 7(4), 10.1111/jicd.12176 (2016).
Heavy smokers showed a trend toward higher pathogen loads (T. forsythia, T. denticola, P. gingivalis) and elevated cytokines, though differences were not statistically significant in healthy peri-implant tissues.

Tomišič, K., Rodič, K., Sotošek, A., et al. Do Differences in Cultivable Subgingival Species Exist between Different Periodontitis Stages and Grades? Oral Health Prev. Dent. 19(1), 10.3290/j.ohpd.b875525 (2021).
F. nucleatum counts were significantly higher in grade B than grade C periodontitis, while other cultivable species, including P. gingivalis and A. actinomycetemcomitans, showed similar prevalence across stages and grades.

Soldati, K. R., Gutierrez, L. S., Anovazzi, G., Scarel-Caminaga, R. M., & Zandim-Barcelos, D. L. Impact of smoking on protein levels of beta-defensins in periodontal disease. Brazilian Dental Journal 33(4), 10.1590/0103-6440202204685 (2022).
This study investigated how smoking affects beta-defensin (hBD) 1 and 2 protein levels in periodontitis patients. Gingival crevicular fluid (GCF) analysis revealed that smokers had reduced hBD 1 and increased hBD 2 in diseased sites. Healthy sites from non-periodontitis patients exhibited higher hBD 1 levels, indicating that smoking may impair antimicrobial peptide regulation and contribute to periodontal disease progression.

Guglielmetti, M. R., Rosa, E. F., Lourenço, D. S., Inoue, G., Gomes, E. F., De Micheli, G., Mendes, F. M., Hirata, R. D. C., Hirata, M. H., & Pannuti, C. M. Detection and quantification of periodontal pathogens in smokers and never-smokers with chronic periodontitis by real-time polymerase chain reaction. Journal of Periodontology 85(10), 10.1902/jop.2014.140048 (2014).
Smokers with chronic periodontitis exhibited significantly higher counts of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia compared to never-smokers. This study emphasizes the impact of smoking on the subgingival microbiota and its potential role in exacerbating periodontal tissue destruction.

Field, C. A., Gidley, M. D., Preshaw, P. M., & Jakubovics, N. Investigation and quantification of key periodontal pathogens in patients with type 2 diabetes. Journal of Periodontal Research 47(4), 10.1111/j.1600-0765.2011.01455.x (2012).
Quantitative PCR analysis demonstrated that Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum levels differed according to periodontal health but not significantly between type 2 diabetic and nondiabetic subjects. The study suggests that periodontal disease status rather than diabetes per se primarily determines subgingival microbial composition.

Bechir, E. S. The clinical and microbiological effects of LANAP compared to scaling and root planing alone in the management of periodontal conditions. Diagnostics 13(14), 10.3390/diagnostics13142450 (2023).

The laser-assisted new attachment procedure (LANAP) reduced periodontal pocket depth and Porphyromonas gingivalis load more effectively than conventional scaling and root planing. LANAP showed improved clinical outcomes, including reduced bleeding on probing, suggesting its clinical advantage in managing chronic periodontitis.

Li, Z., Chen, S., Liu, C., Zhang, D., Dou, X., & Yamaguchi, Y. Quantification of periodontal pathogens cell counts by capillary electrophoresis. Journal of Chromatography A 1361, 10.1016/j.chroma.2014.07.100 (2014).
Capillary electrophoresis was applied to quantify Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in gingival crevicular fluid. The method provided rapid and accurate measurement of pathogen cell counts, demonstrating a novel tool for monitoring periodontal pathogen dynamics during orthodontic treatment.

Boutaga, K., Savelkoul, P. H. M., Winkel, E. G., & van Winkelhoff, A. J. Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction. Journal of Periodontology 78(1), 10.1902/jop.2007.060078 (2007).
Real-time PCR analysis of subgingival plaque and mouthwash samples showed that oral lavage could detect major periodontal pathogens, including Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, with high sensitivity. The study suggests mouthwash sampling as a rapid, less invasive alternative for periodontal microbial assessment.

Hatanaka, K., Shirahase, Y., Yoshida, T., Kono, M., Toya, N., Sakasegawa, S. I., Konishi, K., Yamamoto, T., Ochiai, K., & Takashiba, S. Enzymatic measurement of short-chain fatty acids and application in periodontal disease diagnosis. PLoS ONE 17(7), 10.1371/journal.pone.0268671 (2022).
An enzymatic assay for short-chain fatty acids in gingival crevicular fluid correlated with periodontal disease severity. Elevated levels of propionic, butyric, and valeric acids were associated with periodontitis, providing a potential noninvasive biomarker for disease monitoring.

Umeizudike, K., Räisänen, I., Gupta, S., Nwhator, S., Grigoriadis, A., Sakellari, D., & Sorsa, T. Active-matrix metalloproteinase-8: a potential biomarker of oral-systemic link. Clinical and Experimental Dental Research 8(1), 10.1002/cre2.516 (2022).
Active-matrix metalloproteinase-8 (aMMP-8) may serve as a biomarker linking periodontal and systemic inflammation. Review of recent studies suggests aMMP-8 reflects host-driven pathology, providing a tool for bridging oral and systemic disease evaluation.

Jelihovschi, I., Drochioi, C., Badescu, A. C., et al. Comparison of sampling techniques for qPCR quantification of periodontal pathogens. Revista de Chimie 68(12), 10.37358/rc.17.12.5993 (2017).
Paper point sampling in subgingival plaque can introduce quantification biases due to gingival bleeding. This study compared paper points and curettes for qPCR detection of Aggregatibacter actinomycetemcomitans and Treponema denticola, highlighting sampling effects on pathogen quantification.

Jervøe-Storm, P. M., AlAhdab, H., Koltzscher, M., Fimmers, R., & Jepsen, S. Quantification of periodontal pathogens by paper point sampling from the coronal and apical aspect of periodontal lesions by real-time PCR. Clinical Oral Investigations 14(5), 10.1007/s00784-009-0333-x (2010).
Real-time PCR analysis of paper point samples from different depths of periodontal lesions revealed comparable recovery of six key pathogens. Total bacterial counts were higher in full-length samples, but correlations suggest both sampling approaches reliably quantify periodontal microbiota.

Herrera, B. S., Henz, S. L., Dua, S., et al. Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT). Archives of Oral Biology 152, 10.1016/j.archoralbio.2023.105721 (2023).
The ROQT method enabled sensitive detection of both cultivated and uncultivated periodontal taxa. Severe periodontitis samples contained high levels of Tannerella forsythia, Porphyromonas gingivalis, and newly recognized pathogens like Filifactor alocis, providing a comprehensive tool for pathogen profiling.

Kanoute, A., Gare, J., Meda, N., et al. Effect of oral prophylactic measures on the occurrence of pre-eclampsia (OP-PE) in high-risk pregnant women: a cluster randomized controlled trial. Methods and Protocols 4(3), 10.3390/mps4030061 (2021).
Implementation of oral hygiene interventions in high-risk pregnant women reduced periodontal pathogen load and inflammation. This cluster randomized trial aims to link oral dysbiosis modulation with decreased risk of pre-eclampsia.

Van der Weijden, F., Rijnen, M., & Valkenburg, C. Comparison of three qPCR-based commercial tests for detection of periodontal pathogens. Scientific Reports 11(1), 10.1038/s41598-021-85305-3 (2021).
Three commercial qPCR kits showed high but slightly varying accuracy in detecting key periodontal pathogens. Kit A detected pathogens more frequently and in higher counts, emphasizing the need to consider test selection in clinical decision-making.

Lages, E. J. P., Costa, F. O., Cortelli, S. C., et al. Alcohol consumption and periodontitis: quantification of periodontal pathogens and cytokines. Journal of Periodontology 86(9), 10.1902/jop.2015.150087 (2015).

Alcohol dependence was associated with higher levels of Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum, along with elevated IL-1β. Findings suggest alcohol negatively influences periodontal microbiology and inflammation.

Takahashi, K., Cunha, R. F., & Jardim, E. G. Periodontal pathogen colonization in young children by PCR quantification—a longitudinal survey. Journal of Clinical Pediatric Dentistry 41(6), 10.17796/1053-4628-41.6.7 (2017).
Longitudinal analysis revealed early colonization of children by maternal periodontal pathogens. Mother-child microbial correlation suggests early biofilm control may prevent acquisition of aggressive species.

Kirakodu, S. S., Govindaswami, M., Novak, M. J., Ebersole, J. L., & Novak, K. F. Optimizing qPCR for the quantification of periodontal pathogens in a complex plaque biofilm. The Open Dentistry Journal 2(1), 10.2174/1874210600802010049 (2008).
This study developed a standardized qPCR protocol for mixed plaque samples, ensuring reproducibility and accurate quantification of specific periodontal bacteria in complex biofilms.

Zhou, X., Liu, X., Li, J., et al. Real-time PCR quantification of six periodontal pathogens in saliva samples from healthy young adults. Clinical Oral Investigations 19(4), 10.1007/s00784-014-1316-0 (2015).
Real-time PCR enabled quantification of six periodontal pathogens in saliva from healthy adults. Fusobacterium nucleatum was most abundant, demonstrating saliva as a practical diagnostic fluid for monitoring subgingival pathogens.

Coffey, J., Choudhry, M., Shlossman, M., Makin, I. R. S., & Singh, V. K. Multiplex real-time PCR detection and relative quantification of periodontal pathogens. Clinical and Experimental Dental Research 2(3), 10.1002/cre2.37 (2016).
Multiplex qPCR assay detected and quantified five key periodontal pathogens simultaneously. This approach provides an efficient tool for profiling periodontal microbiota and can assist clinical research and diagnostics.

Takahashi, K., Cunha, R. F., & Jardim, E. G. Periodontal pathogen colonization in young children by PCR quantification—a longitudinal survey. Journal of Clinical Pediatric Dentistry

42(2), 10.17796/1053-4628-42.2.4 (2018).
Confirming previous findings, this study reinforced that early microbial colonization in children is strongly influenced by maternal oral microbiota, highlighting the importance of early preventive strategies.

Choi, H., Kim, E., Kang, J., et al. Real-time PCR quantification of nine periodontal pathogens in saliva samples from periodontally healthy Korean young adults. Journal of Periodontal and Implant Science 48(4), 10.5051/jpis.2018.48.4.261 (2018).
Saliva from healthy young adults contained a high prevalence of periodontal pathogens, with Fusobacterium nucleatum being the most abundant. This highlights the potential risk of future periodontal disease even in clinically healthy individuals.

Decat, E., Cosyn, J., De Bruyn, H., et al. Optimization of quantitative polymerase chain reactions for detection and quantification of eight periodontal bacterial pathogens. BMC Research Notes 5, 10.1186/1756-0500-5-664 (2012).
Optimized qPCR protocols were developed for eight key periodontal pathogens and Streptococcus mutans, providing reliable quantification for assessing periodontal risk and monitoring therapeutic outcomes.

Torrungruang, K., Chantarangsu, S., Sura, T., & Thienpramuk, L. Interplay between vitamin D receptor FokI polymorphism and smoking influences Porphyromonas gingivalis proportions in subgingival plaque. Journal of Clinical Periodontology 47(8), 10.1111/jcpe.13307 (2020).
The VDR FokI polymorphism interacted with smoking to increase subgingival Porphyromonas gingivalis levels and periodontitis severity. Host genetics combined with lifestyle factors can significantly influence periodontal microbial composition and disease risk.