ADVANCES IN PERIODONTAL DIAGNOSTIC TESTING

Post-Graduate Theory Examination Answer (50 Marks)

PART I: CLASSIFICATION OF ADVANCES IN PERIODONTAL DIAGNOSTIC TESTING

A. ADVANCES IN ASSESSMENT OF GINGIVAL INFLAMMATION

1. Gingival temperature measurement - Subgingival temperature is elevated at sites with active periodontal disease; measured using temperature probes.
2. Gingival crevicular fluid (GCF) volume - Increased GCF volume correlates with gingival inflammation; measured using Periotron device.
3. Tissue oxygen tension - Reduced at inflamed sites; measured by polarographic electrodes.
4. Bleeding on probing (BOP) - Standardized quantification using pressure-sensitive probes.
5. Laser Doppler Flowmetry - Measures microvascular blood flow in gingival tissue.

B. ADVANCES IN ASSESSMENT OF LOSS OF PERIODONTAL ATTACHMENT

1. First generation probes - Conventional manual probes (Williams, UNC-15).
2. Second generation probes - Constant force / pressure-sensitive probes (e.g., Florida Probe) that standardize probing force at 25 g.
3. Third generation probes - Computerized probes with electronic data capture.
4. Fourth generation probes - Probes utilizing 3-dimensional (3D) technology.
5. Fifth generation probes - Probes utilizing 3D technology combined with ultrasound.

C. ADVANCES IN RADIOGRAPHIC DIAGNOSIS

1. Digital radiography - Direct (Charge-Coupled Device / CCD; Complementary Metal Oxide Semiconductor / CMOS) and indirect (storage phosphor / photostimulable plate) systems.
2. Subtraction radiography - Detection of sequential bone density changes.
3. Digital subtraction radiography (DSR) - Software-based pixel subtraction; introduced by Webber (1982) and Grondahl (1983).
4. Computer Assisted Densitometric Image Analysis (CADIA) - Quantitative bone density analysis.
5. Cone Beam Computed Tomography (CBCT) - 3D volumetric imaging of alveolar bone.
6. Magnetic Resonance Imaging (MRI) - Non-ionizing modality gaining momentum (Yeung et al., 2024).
7. Quantitative ultrasound - Bone quality assessment.

D. ADVANCES IN MICROBIOLOGICAL ANALYSIS

1. Bacterial culturing
2. Microscopic identification (light, dark-field/phase contrast, fluorescence)
3. Chromatography (High-Performance Liquid Chromatography / HPLC)
4. Immunodiagnostic techniques
5. Molecular biology techniques
6. Next-generation sequencing (NGS) / Metagenomics
7. Metatranscriptomics and metaproteomics

E. ADVANCES IN IMMUNODIAGNOSTIC TECHNIQUES

1. Immunofluorescence assays - Direct and indirect; antibodies conjugated to fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC).
2. Radio-immunoassay (RIA) - Radiolabeled antigen-antibody reactions; detects nanomolar/picomolar concentrations.
3. Enzyme-Linked Immunosorbent Assay (ELISA) - Gold standard; Evalusite kit (Eastman Kodak) detects P. gingivalis, P. intermedius, A. actinomycetemcomitans within 8 minutes chairside.
4. Flow cytometry - Measures particle characteristics in fluid stream using laser detection.
5. Latex agglutination assays - Rapid chairside tests; antibody-coated latex particles.
6. Immunoblotting / Western blotting - Determines molecular weight and quantity of antigens.

F. ADVANCES IN MOLECULAR BIOLOGY TECHNIQUES

1. Nucleic acid hybridization - Whole genomic DNA probes; Checkerboard DNA-DNA Hybridization.
2. Nucleic acid amplification - Polymerase Chain Reaction (PCR) and its variants.
3. Nucleic acid sequencing - 16S ribosomal RNA (rRNA) gene sequencing; Shotgun metagenomics.
4. Enzymatic digestion of nucleic acids - DNA fingerprinting using restriction endonucleases.

G. ADVANCES IN BIOMARKER IDENTIFICATION (GCF Analysis)

1. Subgingival bacteria and their products
2. Inflammatory and immune products (cytokines, immunoglobulins)
3. Proteolytic and hydrolytic enzymes (Matrix Metalloproteinases / MMPs, elastase, beta-glucuronidase)
4. Enzymes released from dead cells (aspartate aminotransferase / AST, lactate dehydrogenase / LDH)
5. Connective tissue degradation products (hydroxyproline, glycosaminoglycans)

H. ADVANCES IN GENETIC TESTING

1. Interleukin-1 (IL-1) genotype test (Periodontal Susceptibility Test / PST).
2. Genetic polymorphism testing for cytokine genes.

I. ADVANCES IN CHAIRSIDE DIAGNOSTIC TESTS

1. Evalusite (Eastman Kodak) - ELISA-based, detects 3 pathogens in 8 minutes.
2. BANA test (Benzoyl-DL-Arginine Naphthylamide) - Detects trypsin-like enzymes from T. denticola, P. gingivalis, T. forsythia.
3. Perio 2000 Sulfide Sensor - Measures volatile sulfur compounds (VSC) produced by periodontal pathogens.
4. PerioPOC® and MyPerioPath® - Commercial quantitative PCR (qPCR) chairside panels.

PART II: ADVANCES IN MICROBIOLOGICAL ANALYSIS - DETAILED DESCRIPTION

1. INTRODUCTION AND RATIONALE

• The oral cavity harbors 774 species-level taxa in homeostatic balance; disruption causes dysbiosis, which precedes periodontal tissue destruction. (Manoil et al., 2024)
• Conventional diagnosis using probing depth (PD), clinical attachment level (CAL), and radiographs only reflects historic damage - they cannot detect active or impending destruction.
• Socransky et al. (1970s-80s) revolutionized the field by showing that attachment loss occurs in random bursts (active/quiescent phases), creating the impetus for real-time microbial diagnostics.
• The aim of microbiological analysis is to: identify causative organisms, guide antimicrobial therapy, monitor treatment, and predict disease recurrence.

2. BACTERIAL CULTURING

Principles

• Considered the mainstay of phenotypic bacterial characterization. (Manoil et al., 2024)
• Organisms are grown in aerobic or anaerobic conditions on selective/differential media.
• Identifies bacteria by morphology, metabolic capability, nutritional requirements, and antibiotic susceptibility.

Phenotypic Criteria of Identification:

• Microscopic morphology and staining (Gram stain)
• Colony (macroscopic) morphology
• Environmental requirements for growth (aerobic, facultative, obligate anaerobe)
• Resistance/susceptibility to antimicrobials
• Nutritional requirements and metabolic capabilities

Antimicrobial Susceptibility Testing (AST):

• Clinical bacterial isolates are plated on agar and exposed to antibiotics via disc/gradient strip diffusion.
• Determines Minimum Inhibitory Concentration (MIC) values.
• Clinical breakpoints established by EUCAST (European Committee on Antimicrobial Susceptibility Testing) and CLSI (Clinical Laboratory Standards Institute).
• An isolate with MIC below the breakpoint = "susceptible"; above = "resistant."
• AST is the only tool to confirm phenotypical antibiotic resistance unequivocally.
• Studies from Spain and Netherlands showed increased resistance of A. actinomycetemcomitans, F. nucleatum, and P. intermedia to amoxicillin in Spanish isolates; 54% of Prevotella spp. produced beta-lactamase. (Manoil et al., 2024)

Limitations of Bacterial Culturing:

• Time-consuming and expensive.
• Requires viable (live) bacteria.
• ~30% of subgingival taxa remain unculturable (e.g., Treponema spp., certain Bacteroides).
• Fastidious organisms require specific transport conditions.
• Results affected by sampling method and transport.

3. MICROSCOPIC IDENTIFICATION

a. Dark-Field Microscopy

• Creates bright image against dark background.
• Assesses morphology and motility of bacteria in plaque samples directly.
• Evaluated systematically by Listgarten & Hellden (1978).
• Advantages: Inexpensive, rapid, enables visualization of major morphotypes (spirochetes, motile rods).
• Disadvantages:
  • Cannot identify individual species.
  • Major pathogens (A. actinomycetemcomitans, P. gingivalis, T. forsythia, Eikenella corrodens, Eubacterium spp.) are non-motile - not detectable.
  • No guidance for antibiotic selection.
  • Studies could not establish correlation between spirochetal/motile forms and disease progression in maintenance patients.

b. Phase Contrast Microscopy

• Objects differing slightly in refractive index or thickness are distinguished from unstained/living cells.
• Advantage over dark-field: Visualizes cell organelles and structures otherwise invisible.
• Differential retardation of light shifts the phase, producing contrast.

c. Fluorescence Microscopy

• Fluorochrome excited by ultraviolet (UV) light produces visible fluorescence.
• Fluorescence emission is always a longer wavelength (less energy) than excitation (Stokes' shift).
• Produces bright image on dark background.
• Used to study:
  • Intracellular distribution and dynamics of macromolecules
  • Specific organelles, filaments, membrane regions
  • Immunofluorescence identification of specific bacteria

4. CHROMATOGRAPHY

• Periodontal pathogens (T. denticola, P. gingivalis, P. intermedia, T. forsythia) degrade serum proteins, cysteine, and methionine, producing Volatile Sulfur Compounds (VSC): H₂S (hydrogen sulfide), CH₃SH (methyl mercaptan).
• High-Performance Liquid Chromatography (HPLC):
  • Separates and quantifies compounds dissolved in solution.
  • Used to identify bacterial cell wall components and molecules in antibiotic-resistant bacteria.
  • Identifies metabolites and VSC produced by pathogens.
• Perio 2000 Sulfide Sensor (Diamond General Corp.): measures sulfide compounds on a scale of 0-5 (score 5 = 0.1 M sulfide); non-specific but predicts periodontal pathogen presence.

5. IMMUNODIAGNOSTIC TECHNIQUES

a. Immunofluorescence Assays

• Fluorescent-labeled antibodies (FITC or TRITC) detect specific bacterial antigens.
• Direct IF: Single labeled primary antibody directed against the target antigen.
• Indirect IF: Unlabeled primary antibody + fluorophore-conjugated secondary antibody.
• Detection using flow cytometer, array scanner, or fluorescence microscopy.

b. Radio-Immunoassay (RIA)

• Radiolabeled antigen competes with unlabeled sample antigen for antibody binding sites.
• Detects nanomolar and picomolar concentrations of substances.
• Radioactive isotopes: ¹²⁵Iodine most commonly used.
• First immunoassay technique developed; highly sensitive and specific.

c. Enzyme-Linked Immunosorbent Assay (ELISA)

• Gold standard of clinical immunodiagnostics.
• Used primarily in periodontology to detect serum antibodies to periodontopathogens.
• Antigen immobilized on polystyrene microtiter plate wells (96-well or 384-well).
• Sandwich ELISA: analyte bound between two primary antibodies (capture + detection antibody) - most sensitive format.
• Enzyme conjugate (horse-radish peroxidase) cleaves colorless substrate to colored product.
• Evalusite (Eastman Kodak): Chairside ELISA kit; detects P. gingivalis, P. intermedius, A. actinomycetemcomitans within 8 minutes; results read on semiquantitative color intensity scale.

d. Flow Cytometry

• Measures physical and chemical characteristics (size, granularity, fluorescence) of particles in fluid stream using laser beams.
• Three components: fluidics, optics, electronics.
• Applied for identification of oral bacteria; technical standardization challenges remain.

e. Latex Agglutination Assays

• Antibody-coated latex particles agglutinate visibly in the presence of specific antigens.
• Indirect assay: Antibody bound to latex; mixed with plaque sample; agglutination = positive result.
• Inhibition assay: Fixed antibody + test sample; degree of inhibition of carrier particle aggregation indicates antigen concentration.

6. CHECKERBOARD DNA-DNA HYBRIDIZATION

• Introduced by Socransky in the 1990s; a landmark technique. (Manoil et al., 2024; Socransky et al., 1998)
• Modified Southern blot technique enabling detection of up to 40 bacterial species in a maximum of 28 clinical samples on one nylon membrane.
• Uses whole-genomic DNA probes - purified DNA extracts from pure cultures.
• Co-occurring species clusters statistically associated with disease severity.
• Led to the classification of subgingival bacteria into 5 color complexes:
  • Red complex (P. gingivalis, Tannerella forsythia [formerly Bacteroides forsythus], Treponema denticola) - most strongly correlated with deep pockets, BOP, and active disease.
  • Orange complex (Fusobacterium nucleatum/periodonticum, Prevotella intermedia, Prevotella nigrescens, Peptostreptococcus micros, Eubacterium nodatum, Campylobacter rectus, C. showae, Streptococcus constellatus, C. gracilis)
  • Yellow complex (S. sanguis, S. oralis, S. mitis, S. gordonii, S. intermedius)
  • Green complex (3 Capnocytophaga spp., C. concisus, Eikenella corrodens, A. actinomycetemcomitans serotype a)
  • Purple complex (Veillonella parvula, Actinomyces odontolyticus)
• Advantage: Multiple species, multiple samples, no culturing required.
• Limitation: Detection essentially limited to culturable taxa (probes generated from pure cultures).

7. 16S RIBOSOMAL RNA (rRNA) GENE-BASED TECHNIQUES

Background:

• Emerged in 1990 as a revolutionary taxonomic tool.
• 16S rRNA gene (~1500 bp) is ubiquitous in all prokaryotes (encodes small ribosomal subunit).
• Contains 9 variable regions (V1-V9) interspersed with conserved regions.
• Oral microbiology typically targets regions V1-V2, V3-V4, or V4 alone.
• V1-V2 (and V3) regions display higher discrimination potential, distinguishing species within the genus Streptococcus.

a. Closed-Ended (Taxa-Targeted) 16S Application - PCR

• Species-specific primers anneal within variable regions.
• Amplification confirms presence of specific taxa.
• Culture-independent; highly sensitive.

b. Nucleic Acid Amplification - PCR Variants:

• Taq polymerase is the standard enzyme (functions efficiently at 72°C; withstands 94°C denaturation).

c. Commercial qPCR Services:

• IAI PadoTest® (Switzerland/Germany/Europe)
• PerioPOC® (worldwide)
• MyPerioPath® and HR5® (United States)
• Typically quantify panels of 5-11 periodontal taxa classified according to Socransky's color complexes.
• Clinical use: Guide decision on adjunctive antibiotherapy (especially when red complex or A. actinomycetemcomitans detected).
• A. actinomycetemcomitans detection: adjunct amoxicillin or moxifloxacin may suppress the taxon (also invades mucosae beyond pockets, so mechanical debridement alone is insufficient). (Manoil et al., 2024)

8. OPEN-ENDED 16S rRNA COMMUNITY PROFILING (NEXT-GENERATION SEQUENCING)

• PCR primers in the flanking conserved regions amplify any 16S variable region - detects all bacteria in a sample, regardless of prior characterization.
• Unveiled ~400 species-level taxa in the subgingival microbiota, of which 215 were novel phylotypes previously unknown.
• Taxonomy assigned by comparison with databases (e.g., Human Oral Microbiome Database / HOMD); unknown sequences assigned a "phylotype."
• Led to the "Ecological Plaque Hypothesis": microbiota and host coexist in homeostasis; dysbiosis triggers disease, not individual pathogens.
• Bridged the apparent dichotomy between non-specific and specific plaque hypotheses.
• Supports the current model of "Polymicrobial Synergy and Dysbiosis" (Hajishengallis).

Subgingival Microbial Dysbiosis Index (SMDI):

• Machine learning algorithms re-analyzed 16S datasets to identify species-level taxa discriminating health from disease. (Manoil et al., 2024)
• More than 200 differentially abundant species identified; narrowed to 49 "discriminator" species.
• Periodontitis markers: T. denticola, T. forsythia, Mogibacterium timidum, F. alocis, Fretibacterium spp.
• Health markers: S. sanguinis, Actinomyces naeslundii, Veillonella, Neisseria, Rothia, Corynebacterium, Actinomyces.
• SMDI tested longitudinally: significant decrease from baseline to day 1 post-scaling and root planing (SRP), stabilizing up to 3 months.

9. SHOTGUN METAGENOMIC SEQUENCING

• Exploits every fragment of bacterial genome in a sample (not only 16S).
• Generates "reads" from aggregate genomes of the entire community.

Two Analytical Approaches:

1. Read-based analysis (mapping): Reads computed against annotated databases; profiles taxonomy to species level and predicts community metabolism. Less computationally demanding; relies on database comprehensiveness.
2. Assembly-based analysis: Reads reassembled into whole genomes; computationally intensive; can reconstruct genomes of entirely novel organisms with no sequenced relatives.

Clinical Applications:

• Virulence genotyping: Identifies strain-specific variations (e.g., A. actinomycetemcomitans JP2 genotype - 530 bp deletion in ltxA promoter - elevated leukotoxin LtxA expression, significantly elevated risk of periodontitis in young individuals).
• Antibiotic Resistance Gene (ARG) detection: Identifies ARGs irrespective of taxonomic assignment; guides antibiotic selection; surveils emerging resistances.
  • Point-of-care solutions: streamlined qPCR on ready-to-use disks requiring only 200 µL saliva; results within 3 hours. (Manoil et al., 2024)
• Commercial kit: PadoBiom® (IAI, Switzerland): Provides community diversity evaluation, dysbiotic state assessment, red complex + A. actinomycetemcomitans + F. alocis abundance, A. actinomycetemcomitans serotype genotyping (a,b,c,d,e including JP2), ARG detection for 5 antibiotic classes (beta-lactams, nitroimidazoles, tetracycline, quinolones, macrolides).

10. NUCLEIC ACID SEQUENCING AND DNA FINGERPRINTING

DNA Sequencing:

• Determines nucleotide order (adenine, guanine, cytosine, thymine) in a DNA molecule.
• Nucleotide sequence determines amino acid order and hence protein structure (proteomics).
• Sequence of test microorganism compared with known DNA sequences to determine identity.

DNA Fingerprinting (Restriction Fragment Length Polymorphism / RFLP):

• Restriction endonucleases cut chromosomal DNA at specific nucleotide sequences.
• Resultant DNA fragments separated by electrophoresis - produces a unique "fingerprint."
• Strains with similar nucleotide sequences produce amplicons of similar sizes.
• Used to identify, compare, and differentiate bacterial strains (clonal type analysis).

11. METATRANSCRIPTOMICS

• Analyzes the entire set of active gene transcripts (messenger RNA / mRNA) in the microbial community.
• Reveals disease-specific microbial transcripts and a conserved "core metatranscriptome" in periodontitis.
• Core metatranscriptome involves: iron acquisition, lipopolysaccharide (LPS) synthesis, and short-chain fatty acid (SCFA) production.
• Interesting finding: bacteria usually associated with health may exhibit heightened transcriptional activity during periodontitis, expressing virulence factors (cobalamin synthesis, proteolysis, potassium transport).
• Treatment diminishes bacterial activity rather than fully restoring a "healthy" microbiota.

12. METAPROTEOMICS

• Provides a comprehensive, real-time snapshot of the subgingival microbiota by analyzing the entire collection of proteins expressed.
• Tools: Mass spectrometry (MS) + bioinformatics (e.g., liquid chromatography-tandem mass spectrometry / LC-MS/MS).
• Characterizes in vitro mono- or multispecies subgingival biofilms.
• Sheds light on "which organism is doing what" - metabolic interactions and nutrient competition.
• Key finding: Anaeroglobus geminatus (strictly anaerobic gram-negative coccus) instigates proliferation of P. intermedia and up-regulates virulence across the entire community.
• Filifactor alocis secretes extracellular vesicles interacting with Toll-like receptor 2 (TLR-2), promoting osteoclastogenesis.
• Application: still in early stages for oral microbial communities; complexity and heterogeneity pose challenges.

13. BIOMARKERS IN GCF FOR MICROBIOLOGICAL ASSESSMENT

• Gingival Crevicular Fluid (GCF) is the most important source of disease biomarkers.
• For a GCF component to be a valid biomarker: exact source, precise nature, and role in disease must be established.

Categories of GCF Biomarkers:

14. FUTURE DIRECTIONS IN MICROBIAL DIAGNOSTICS

1. Point-of-care (POC) testing combining microbial + host biomarkers for early dysbiosis detection.
2. Early diagnosis before clinical signs - dysbiosis precedes inflammation; microbial biomarkers can predict upcoming destruction.
3. Experimental gingivitis model data: Changes in plaque composition and metabolites detectable within 24 hours of stopping oral hygiene.
4. ARG surveillance to combat global antibiotic resistance - periodic periodontal resistome profiling.
5. Ecological modulation - targeting metabolic networks of periodontal communities rather than individual pathogens.
6. Cumulative biomarker indices (microbial + host) for maximum diagnostic accuracy and prediction of disease relapse during maintenance.
7. Standardized protocols for sample collection, methodologies, and analysis pipelines needed for reproducibility.

SUMMARY TABLE: COMPARISON OF MICROBIOLOGICAL DIAGNOSTIC METHODS

REFERENCES (from uploaded documents)

1. Manoil D, Parga A, Bostanci N, Belibasakis GN. Microbial diagnostics in periodontal diseases. Periodontology 2000. 2024;95:176-193.
2. Saroch N. Advanced diagnostic techniques in periodontics. In: Periobasic: A Text Book of Periodontology and Implant Dentistry. Ch. 43.
3. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent Jr RL. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25:134-144.
4. Curtis M. Periodontal infections. In: Lang NP, Lindhe J (eds). Clinical Periodontology and Implant Dentistry. 6th Ed. Wiley-Blackwell, 2015; Ch. 10.
5. Quirynen M, Teughels W, Haake SK, Newman MG. Microbiology of periodontal diseases. In: Carranza's Clinical Periodontology. 10th Ed. Elsevier, 2006; Ch. 9.
6. Kinane DF, Bornstein MM. Introduction to the Diagnostics in Periodontology and Implant Dentistry issue. Periodontology 2000. 2024;95:7-9.

• GCF = Gingival Crevicular Fluid
• PCR = Polymerase Chain Reaction
• qPCR = Quantitative Polymerase Chain Reaction
• NGS = Next-Generation Sequencing
• ELISA = Enzyme-Linked Immunosorbent Assay
• RIA = Radio-Immunoassay
• FITC = Fluorescein Isothiocyanate
• TRITC = Tetramethylrhodamine Isothiocyanate
• HPLC = High-Performance Liquid Chromatography
• VSC = Volatile Sulfur Compounds
• AST = Antimicrobial Susceptibility Testing
• MIC = Minimum Inhibitory Concentration
• EUCAST = European Committee on Antimicrobial Susceptibility Testing
• CLSI = Clinical Laboratory Standards Institute
• ARG = Antibiotic Resistance Gene
• 16S rRNA = 16S Ribosomal Ribonucleic Acid
• SMDI = Subgingival Microbial Dysbiosis Index
• LC-MS/MS = Liquid Chromatography-Tandem Mass Spectrometry
• CBCT = Cone Beam Computed Tomography
• CADIA = Computer Assisted Densitometric Image Analysis
• DSR = Digital Subtraction Radiography
• BOP = Bleeding On Probing
• CAL = Clinical Attachment Level
• PD = Probing Depth
• BANA = Benzoyl-DL-Arginine Naphthylamide
• TLR-2 = Toll-Like Receptor 2
• LPS = Lipopolysaccharide
• SCFA = Short-Chain Fatty Acids
• MMP = Matrix Metalloproteinase
• LDH = Lactate Dehydrogenase
• IL = Interleukin
• TNF-α = Tumor Necrosis Factor-alpha
• HOMD = Human Oral Microbiome Database
• POC = Point-of-Care
• CCD = Charge-Coupled Device
• CMOS = Complementary Metal Oxide Semiconductor
• RT-PCR = Reverse Transcription PCR
• RFLP = Restriction Fragment Length Polymorphism
• SRP = Scaling and Root Planing
• UNC = University of North CarolinaThe answer above has been synthesized entirely from your uploaded references. Here is a brief guide to its structure:

1. Introduction and rationale
2. Bacterial culturing and AST (Antimicrobial Susceptibility Testing)
3. Dark-field, phase contrast, and fluorescence microscopy
4. Chromatography and HPLC (High-Performance Liquid Chromatography)
5. Immunodiagnostic techniques - ELISA, RIA, flow cytometry, latex agglutination
6. Checkerboard DNA-DNA Hybridization and the 5 color complexes (Socransky 1998)
7. 16S rRNA gene techniques and PCR variants (multiplex, nested, real-time, qPCR)
8. Open-ended 16S NGS (Next-Generation Sequencing) and SMDI (Subgingival Microbial Dysbiosis Index)
9. Shotgun metagenomics with ARG (Antibiotic Resistance Gene) detection
10. DNA sequencing and fingerprinting
11. Metatranscriptomics
12. Metaproteomics
13. GCF biomarkers table
14. Future directions