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![OMNIGENE (DMDx) IAI PADO TEST 4.5
Commercially available kit - probes for Pg, Aa, Tf, Td, &Pi. [Eick S Pfischer 2002]
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Uploaded byDr Fariya Ashraf
Advances in Microbiological diagnosis of Periodontal diseases
Dr. Fariya Ashraf presented on advanced microbiological diagnostic aids for periodontal diseases. Various methods were discussed including bacterial culturing, direct microscopy, immunodiagnostic methods like ELISA, enzymatic methods like the BANA test, and new molecular technologies like DNA probes and PCR. Each method has advantages and limitations for identifying putative periodontal pathogens and monitoring periodontal disease and treatment. Molecular methods now allow rapid identification of multiple bacterial species from a single sample and have greater sensitivity than traditional culturing techniques.
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Advances in Microbiological diagnosis of Periodontal diseases
- 1. Presented By Dr.Fariya Ashraf Deptof Periodontology and Oral Implantogy
- 2. Introduction Advanced microbiological diagnostic aids Bacterialculturing Direct microscopy Immunodiagnostic methods Enzymatic methods New molecular technologies- DNA probes & PCR Chair side diagnostic kits Conclusion Reference
- 3. Over 300 bacterialspecies are recognized as being present in the human oral cavity. Only few species are associated with disease… Moore WEC, Moore LVH. The bacteria of periodontal diseases. Periodontol 2000 1994: 5: 66-77. Periodontitis, an infectious disease caused by bacteria, brings about destructive changes leading to loss of bone and connective tissue attachment (Williams, 1990). Several oral bacteria are considered to be possible pathogens in periodontitis (Darveau et al., 1997) INTRODUCTION
- 4. According tocriteria described by Socransky 1) Strong evidence for Aa, Pg, Tf 2) Moderate evidence for campylobacter rectus, eubacterium nodatum, fusobacterium nucleatum, peptostreptococcus micros, prevotella intermedia, prevotella nigrescens, streptococcus intermedius, and spirochetes such as treponema denticola
- 5. 1) To identifyputative pathogens 2) To serve as indicators of disease initiation and progression and healing 3) To determine which periodontal sites are at higher risk for active destruction 4) To monitor periodontal therapy 5) To aid in treatment planning
- 6. Culture method Microscopic Methods Enzymatic method Immunodiagnostic method Diagnostic Assays based upon molecularbiology technique Microbiological Examination Methods for Periodontal Diseases
- 7. Considered as a“Reference method/Gold standard” when determining the performance of new microbiological diagnostic aids BACTERIAL CULTURING Originally used by Louis Pasteur. He used urine and meat broth. First solid medium...cooked cut potato used by Robert Koch
- 8. SOLID MEDIUM SEMISOLID MEDIUM LIQUID MEDIUM
- 9. Steps involved Specimen collection Transport Inoculation Incubation of growth media Identificationof the isolated organisms.
- 10. DISADVANTAGES -Can grow livebacteria only -Requires strict sampling & transport conditions -Some pathogens are fastidious & difficult to grow. -Low senstivity -Time consuming & expensive ADVANTAGES -Can obtain relative & absolute counts of cultured species. -It is the only in vitro method to assess antibiotic susceptibility of microbes.
- 12. PHASE CONTRAST MICROSCOPY ‘Darkfieldor phase contrast microscopy’ – alternative to culture methods. Dark Field Microscopy is a technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.
- 13. To view aspecimen in dark field, an opaque disc is placed underneath the condenser lens, so that only Light that is scattered by objects on the slide can reach the eye . Instead of coming up through the specimen, the light is reflected by particles on the slide. Everything is visible regardless of color, usually bright white against a dark background Ability to directly and rapidly assess the morphology and motility of bacteria in a plaque sample
- 14. Advantages • Directly andrapidly assess the morphology and motility of bacteria • Used to indicate periodontal disease status and to structure maintenance programs. Disadvantages • Unable to identify non motile pathogens • Unable to differentiate among the various species of Treponema.
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- 18. The organism unable tocultivate on artificial media. The organism failed to survive transport to the laboratory To detect individual bacterial species To detect presence & relative proportions of selected bacterial species. INDICATIONS FOR IMMUNODIAGNOSTIC METHOD
- 19. • Used todetect bacterial species that are genetically distinct. They help in identifying target bacteria. • Immuno fluorescence is a process in which dyes called fluorochromes are exposed to UV, violet, or blue light to make them fluorescence or emit visible light. Two types DIRECT IMMUNOFLUORESCENCE INDIRECT IMMUNOFLUORESCENCE
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- 21. • Can beused for identification of bacteria, viruses or other antigens • Pathogens can be identified and quantified by this method by visualizing under a microscope
- 22. Indirect IFA employsa secondary fluorescein – conjugated antibody that reacts with the primary antigen – antibody complex. Mainly detect :A.actinomycetemocomitans and P. gingivalis.
- 23. Zambon et al.(1985) showed this technique was comparable with bacterial culture in its ability to identify Aa and Pg in subgingival plaque sample. Comparative studies indicate that the sensitivity of these assays ranges from 82% to 100% - A. a 91% to 100% for P. gingivalis. specificity values of 88% - 92% -A.a 87% - 89% - P.g respectively. (Zambon 1985, Zambon et al. 1985, 1986) IFA demonstrated – a higher sensitivity when compared with culture, probably because of a lower detection limit.
- 24. • Used forrapid identification of oral bacteria • Highly sophisticated instrument • Very costly
- 26. • Radio isotopesused as labels • Binder-ligand assay • Substance whose concentration is to be determined is termed as analyte or ligand • The binding protein which binds to the ligand is called the binder Can be used for quantitation of hormones, drugs, tumour markers, viral antigens and IgE
- 28. • Enzymes usedas labels • Very versatile, sensitive, simple and economic procedure • Absence of radiation hazard • Test kits available • Two basic types 1)homogeneous 2)heterogeneous
- 29. HOMOGENOUS EIA Noneed to separate the bound and free fractions The test can be completed in one step with all the reagents added simultaneously Used only for haptens such as drugs Eg-Enzyme multiplied immunoassay technique (EMIT) to detect drug molecules like opiates, cocaine, barbiturates, amphetamine in serum
- 30. HETEROGENOUS EIA Requiresseparation of free and bound fractions either by centrifugation or by absorption on solid surfaces and washing. Multistep procedure Reagents added sequentially Eg- enzyme linked immunosorbent assay (ELISA)
- 31. Performing an ELISAinvolves at least one antibody with specificity for a particular antigen. These methods have shown a higher sensitivity and specificity than bacterial culturing for the detection of target microorganisms (Aa, Pg and Tf) ENZYME LINKED IMMUNOSORBENT ASSAY/ELISA TYPES OF ELISA 1. Non Competitive Direct ELISA Indirect ELISA Sandwich ELISA 2. Competitive and Inhibition ELISA
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- 37. The aim ofthe present study was to investigate the association between the serum and salivary antibody levels to A. actinomycetemcomitans and therefore, to find whether this association was varying in different grades of periodontitis. Total of 50 periodontally healthy and 50 chronic periodontitis subjects (35–65 years) of both sexes were included for the study.
- 39. • To detectmicrobial and viral infections, autoimmune diseases, hormones, drugs or serum proteins by agglutination reaction of antigen-antibody. AGGLUTINATION Slide agglutination Latex agglutination DIRECT INDIRECT REVERSE PASSIVE
- 41. Diagnostic assays basedon molecular biology techniques
- 42. Probes consists ofsingle stranded nucleic acid, from a specific microorganism artificially synthesized and labelled for its detection when placed with a plaque sample. – May target whole genomic DNA or individual genes. – Types : Labeled DNA probes Cloned probe/ recombinant probe Oligonucleotide probe Riboprobes or labeled RNA probes nucleic Acid Probe Technology
- 43. Whole genomic probes: morelikely to cross- react with non target microorganisms due to presence of homologous sequences between different bacterial species • Used for the detection of A.a, Pg, Pi and Td Cloned probe /recombinant probe • Sequence of interest & ability to propagate bacteria forms recombined molecule Oligonucleotide probes : Short stretches of single stranded DNA orRNA Display limited or no cross – reactivity with non- target microorganisms. Riboprobes or labeled RNA probes • Are made using invitro transcription systems • 16s rRNA genes contain both regions shared by different bacteria and short stretches of variable regions shared only by specific organisms of the same species or genus.
- 44. PROCESSING The radioactive labels createspots on the film, which are read with a densitometer. The darkness and size of the spots indicate the concentration of the organisms present in the given plaque sample If complementary base pairs hybridize (cross-link), the radiolabeled strands will also be fixed to the filter paper. After the filter is washed to remove any unhybridized strands, it is covered with a radiographic plate.
- 45. OMNIGENE (DMDx) IAIPADO TEST 4.5 Commercially available kit - probes for Pg, Aa, Tf, Td, &Pi. [Eick S Pfischer 2002] MicroDent
- 46. Savitt et al1988, compared the DNA probe with culture method for the detection of A.a, Pg and Pi and reported 100% effectivness in their detection.
- 48. These DNA fragmentpatterns constitute a specific “fingerprint” to characterize each strain The genetic heterogeneity and homogeneity of strains can then be evaluated by comparing the number and size (electrophoretic pattern) of the DNA fragments obtained. The DNA fragments generated are separated by electophoresis, stained with ethidium bromide, and visualized with ultraviolet light. Endonucleases recognize and cleave double – stranded DNA at specific base pair sequences.
- 49. REA is thus– powerful tool for determining the distribution of a specific pathogenic strain through out a population. Also used in molecular genetic analysis of diverse oral bacteria like A.a., P.g.,P.i., E.c.,F.n.,T.d., and has been very useful in studying the transmission patterns of putative periodontal pathogens among family members.
- 51. The most importantcontribution of FISH - positive proof that the oral microorganism community is not randomly distributed but is organized in highly structured biofilms with a distinct architecture (Zijnge et al., 2010) Fluorescence in situ hybridization (FISH)
- 53. ADVANTAGES • Rapid technique •Efficiency of Hybridization and deletion is high. • Sensitivity and specificity is high.. • Cytogenetic data can be obtained from poor samples that contain too few cells for routine cytogenetic analysis. DISADVANTAGES • Practical performance of FISH requires an experienced and highly trained individual. • Its sensitivity is far below the sensitivity of PCR in case of analyzes from primary materials. • The FISH method is not widely commercially available.
- 54. 2018 Journal ofthe International Clinical Dental Research Organization | Published by Wolters Kluwer - Medknow
- 55. DNA Checkerboard isan established technique that gives a simultaneous and quantitative analysis of up to 28 plaque samples against 40 microbial species (Socransky et al., 1994). The assay uses whole genomic, digoxingenin based-labelled DNA Probes and aids in rapid processing of large numbers of plaque samples with multiple hybridization in a single test. The DNA probes used are adjusted to permit the detection of 104 cells of each species. This assay has not been generalized for diagnostic purpose CHECKERBOARD DNA-DNA HYBRIDIZATION TECHNOLOGY
- 58. Advantages • Rapid processingof large no. of plaque sample • Help in identification of multiple bacterial species. • Highly specific and don’t need bacterial viability. • Used for epidemiological research and ecologic studies. • Entire sample may be employed without dilution or amplification thus overcoming problems in encountered in PCR amplification procedures • Recently, checkerboard hybridization has also been used for the quantification of multiple inflammatory mediators in gingival crevicular fluid samples Disadvantages Technique can detect only species for which DNA probes have been prepared. Method requires sophisticated equipment & expertise
- 61. Used almost universallyto study DNA and RNA obtained from a variety of tissue sourses POLYMERASE CHAIN REACTION (PCR) Has emerged as the most powerful tool for the amplification of genes and their RNA transcripts PCR allows large quantities of DNA to be obtained in a simplified and automated manner
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- 63. Average time foreach cycle is approximately 4-5 minutes
- 64. Post amplification detection FollowingPCR, the amplification product can be detected using gel electrophoresis followed by ethidium bromide staining and visualization with UV transillumination. Visualization of a band containing DNA fragments of a particular size - indicate – the presence of the target sequence in the original DNA sample. Absence of a band- indicate – the target sequence was not present in the original DNA sample.
- 65. – ADVANTAGES - –PCR has enabled identification of unculturable bacteria, including spirochetes, that cannot be grown in artificial culture. – PCR helps in monitoring of antibiotic resistance genes during periodontal treatment and thus measuring the effect of antibiotic therapy. – DISADVANTAGES - – Highly sensitive – DNA contamination during processing is a major problem that will also lead to false-positive results.
- 68. – Approach forthe detection of selected bacterial species - to look for the presence of an enzyme that is unique to one or more of the clinically relevant species. ENZYMATIC TESTS
- 69. The BANA testwas developed by - Dr. Walter Löesche and coworkers at Michigan University, being the result of more than 15 years of research Socransky and Haffajee (1987) in their extensive study involving over 10,000 plaque samples taken from over 100 patients, found the BANA positive species, T. denticola, P. gingivalis, and B. forsythus to have the highest prevalence and to be present in the highest levels compared to over 40 other plaque species that were evaluated by DNA probes Hydrolyzed by a trypsin-like enzyme produced primarily by Treponema denticola, Bacteroides forsythus and Porphyromonas gingivalis. BENZOYL ARGININE NAPHTHYLAMIDE (BANA) TEST
- 70. Bretz W, LoescheW. Characteristics of TrypsinLike Activity in Subgingival Plaque Samples,.J. Dent. Res. 1987; 66: 1668-1672 B. forsythus, P. gingivalis, T.denticola, and Capnocytophaga share a common enzymatic profile trypsin-like enzyme This Enzyme activity can be measured with the hydrolysis of the peptide analog/colorless substrate N – benzoyl – arginine – 2 naphthylamide (BANA) Upon hydrolysis, it releases chromophore B- naphthylamide, It turns orange red when a drop of fast garnet is added to the solution Principle of BANAtest
- 71. PERIOSCAN Diagnostic kit (Perioscan®)has been developed using the BANA hydrolysis reaction to detect bacterial trypsin like proteases in the dental plaque. Loesche et al (1999) – proposed the use of BANA reaction in subgingival plaque samples to detect the presence of any of these periodontal pathogens – serve as a marker of disease activity. Beck et al (1995) -used BANA test as a risk indicator for periodontal attachment loss suggested that positive BANA findings - good indication- for presence of T. denticola, P. gingivalis, or both are present at sampled sites
- 72. Positive reaction- Indicatorof presence of pathogens strongly associated with disease when compared to culture, which cannot identify T.d & T.f , Rapid & inexpensive method The BANA enzyme is stable & can be detected in frozen plaque samples. Rapid chairside test with a result in 15 mins It does not include inhibitors of host proteinases which could contaminate the plaque sample from saliva & GCF & which also cleave the BANA substrate False positive results. Detects only 3 pathogens. Inability to determine which of the 3 bacteria are responsible for the enzyme production Does not detect sites undergoing active destruction Advantages Disadvantages
- 74. CHAIR SIDE TESTKITS
- 76. This chairside immunoassay detects periodontal pathogens such as Aa, Pg, Pi. Principle: linkage between the antigen and a membrane bound antibody to form an immunocomplex that is later revealed through a colorimetric reaction when a colored enzyme substrate is added. The main drawback of this test was that it had only been designed to detect 3 out of the dozen or so bacterial species implicated in the pathogenesis of periodontal disease. Positive test forms a blue dot on the reagent pad. Has a detection limit of 105 for Aa and 106 for Pg.
- 77. OMNIGENE These are DNAprobe systems for a number of known periodontopathogen subgingival bacteria. OmniGene Diagnostics, Inc. has applied the principles of genetic engineering to develop species-specific DNA probe tests for eight periodontal pathogens. P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, E. corrodens, C. rectus, B. forsythus, and T. denticola
- 78. IAI Pado Test4.5 – With the Pado RNA probe test kit, four periodontal pathogens can be detected: Aa, Pg, Tannerella Forsythia, and T. denticola. – This test basically uses oligonucleotide probes complementary to conserved fragments of the 16S rRNA gene that encodes the rRNA, which forms a subunit of the bacterial ribosome. The detection frequencies found with this test indicated a low sensitivity of the Pado Test 4.5 method when compared to the checkerboard method.
- 79. Jervoe-storm et al(2005) • Real time PCR for quantitative determination of 6 important marker organisms of Periodontitis and Peri-implantitis (Aa,Pg,Td,Fn,Tf,Pi) • It combines high specificity with high sensitivity and a precise quantification.
- 80. Various methods foridentification of pathogens in periodontal infections Grover, V., Kapoor, A., Malhotra, R., & Kaur, G. (2014). Clinical Relevance of the Advanced Microbiologic and Biochemical Investigations in Periodontal Diagnosis: A Critical Analysis. Journal of Oral Diseases, 2014
- 81. Some retrospectivestudies, have suggested that microbiological assays might be predictive of the clinical course of a site at risk for periodontal breakdown. According to the authors, ". . .these values defined a site with non-progressing disease with 87% sensitivity and 84% specificity." However, they did not state what the corresponding values were for sites with progressing disease. Bragd L et al.1984, Wennström JL et al.1987 Critical levels of the target species were reported to be > 0.01% for A.actinomycetemcomitans; > 0.1% for P.gingivalis; and > 2.5% for P.intermedia.
- 82. REFERENCES • Clinical Periodontologyby Newman 10th Edition. • Clinical Periodontology by Newman 11th Edition. • Periodontology 2000; vol 34; 2004 • Diagnosis & Risk Prediction of Periodontal Diseases Vol 3- Per Axelsson. • Periodontics – Medicine, Surgery and Implants – Rose • Outline of Periodontics - 4th edition JD Manson & Eley
- 83. • Advances inPeriodontics – Wilson, Kornmn & Newmann • Fundamentals of Periodontics- Wilson & Kornmn • Bakermans, C., and E. L. Madsen. 2002. “Detection in Coal Tar Waste-Contaminated Groundwater of mRNA Transcripts Related to Naphthalene Dioxygenase by Fluorescent In Situ Hybridization (FISH) with Tyramide Signal Amplification (TSA),” Journal of Microbiological Methods 50: 75–84. PMID 11943360. • British Dental Journal; vol 184: 1998.