DrFranckChaubron(PhD)CEOIns5tutClinident

Microbiological test systems in periodontology and implantology

Dr. Franck Chaubron

Institut Clinident France

FranckCHAUBRON(PhD),CellularandMolecularBiologistUniversitéBlaisePascal(Fr)-Dundee(UK)CEOofInsGtutClinident(www.insGtut-clinident.com)FranceCEOofInsGtutClinidentBiopharmaSwiss(www.clinidentstemcell.com)SwitzerlandCEOofBlueDNAcompanion(www.bluednacompanion.com)FranceDirectorofBusinessDvpMedXcellS.A(www.medxcell.ch)SwitzerlandPreviousPosiGonManagerandGeneralDirectorofGENOLIFESA(1996–2004)BusinessDeveloperManagerofCAMBREX-LONZA(US-Europe-Japan(2004-December2008)Experiences DentalMedicineandBiology(periodontaldisease,dentalpulpcellbiology,oralcancer)ManagingDirectorofHumanCell&TissueBiobankMicrobiologyMedicaldiagnosGcsGenotyping

CVFranckCHAUBRON

Ourtestpor`oliofororaldiagnosGcsiscurrentlyusedforthefollowingapplicaGons:

•  Periodontaldisease-diagnosis,risk,treatmentdecision

•  Implantology-therapycontrolandimplantmonitoring

•  Cariesdisease-fromsaliva;riskevaluaGon,maintenance•  OralCancer-earlyscreeningfromsalivasample(*2internaGonalpatents)

Technologies:

-MolecularBiology-DNA,RNAquanGtaGverealGme

-Vola5leOrganicCompoundsanalysis-VOCMassSpectrometry

-PointofCare–instrument,realGmePCR

,

INSTITUTCLINIDENTac5vi5es

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A b u n d a n c e

T I C : O C 1 0 0 8 A . D -VOCidenGficaGoninsaliva

-DataBases

- StaGsGc

- DNA&RNA- BacterialDNA

- Y&MDNA

- Virus

MetaboliteBiomarkersanalysis

RealTimePCRDNA/RNABiomarkersanalysis

INSTITUTCLINIDENTDIAGNOSIS/TESTSTECHNOLOGIES

OralTestproductporOolio

RealTimePCRtestbasedonDNAextrac5onandMulGparametricquanGficaGonofDNAbiomarkersandMassMectrometryVOCidenGficaGonTheproposedbiomarkersfororalanddentalapplicaGons•  Bacteria:Aggrega&bacterac&nomycetemcomitans,Porphyromonasgingivalis,Tannerellaforsythensis,Treponemaden&cola,Prevotellaintermedia,Peptostreptococcusmicros,Fusobacteriumnucleatum,Campylobacterrectus,Eikenellacorrodens,Candidaalbicans,Streptococcusspp.,Streptococcusmutans,Streptococcussobrinus,Lactobacillusspp.,Enterococcusspp.,Ac&nomycesviscosus,Capnocytophagaspu&gena,Capnocytophagaochracea,Enterococcusspp.,Enterococcusfaecalis,Ac&nomycesodontoly&cus,Veillonellaparvula,Prevotellanigrescens,Campylobactergracilis,Campylobacterconcisus,Streptococcusmi&s,Streptococcussp.•  CandidaAlbicans•  TotalOralBacteriaCount•  Virus:HPV,Herpes,EBV...•  SalivaVOCiden5fica5oninsaliva•  DentalClinicenvironmentmonitoring:LegionallePneumophila,Speudomonasaeruginosaindentalunitwater,E.coli,Candida,MRSA

Oral Bacterial Etiology

-1970:

Plaque induce periodontitis

1980-1990:

Infection by Anaerobic bacteria

Fusiformis

Steptococci

Spirochetes

Amoeba

FusospirochetalinfecGon

Spirochetes

A.viscosus

A.a.

P.g.

T.f.

1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990

Oral Bacterial Strain History

1year 3years 5years

Periodontaldisease

Mucosi5s Peri-implan55s

Peri-implan55s:InflammatorylesionsthatmayaffectthePeri-ImplantmucosaandmayresultinlossofthesupporGngbonefollowedbydentalimplantfailure

Boneloss Implant Boneloss

From periodontitis to Peri-implantitis

Peri-implantitis Same pathogenic strains as Periodontal disease

Bacterial concentration effect in Peri-implantitis

G.Tabanella, H.Nowzari, J; Slots Clinical Implant Dentistery and Related Research, 11(1), 2009

HigherquanGtyand%

•  28-56%ofpaGentshaveperi-implanGGsatoneormoreimplants.•  (Zitzmann&Berglunch,2008)

Prevalenceofboneloss

•  16%ofpaGents/6.6%oftheimplantshave≥1.8mmbonelossaqer1yearof

implantaGon.(Roos-Jansakeretal.,2006a)

•  28%ofpaGentshaveprogressivebonelossover5yearsof

post-implantaGon.(Franssonetal.,2005)

Prevalence of Peri-implantitis

Haffajee1994

Bonelostin2monthsQuanGtyCFU

Aa Pg

105 3.2mm 2.2mm

106 4.3mm 4.1mm

Bone lost depend of the bacterial identity & quantity in CFU (colony forming unit)

A: Microscopic identification B: Microbiological cultures

Microbiological tests for bacterial identification

C: Nucleic acid probes (PCR)

A: Microscopic Identification Dark field microscopy

E.Cowen,Miscape,1999

B: Microbiological cultures & Identification

RobertKoch,N.Doanetal,JournalofClinicalMicrobiology,1999.

A: GoldstandardmethodB: AllculGvableorganismcanbeidenGfiedC: PhenotypicIdenGficaGonD: AnGbiogram

Bacterial cultures

A:LowleveldetecGonandO2sensiGvityB:NonculGvableorganisms?C:VariaGonbetweenanaerobicculturesystemD:ShortGmeoftransportaGon(24-48h)E:Highcost(labour)F:Prolongedperiodbeforeresults(2weeks)

C: Nucleic acid probes

DNAextractedfromsamplesarehybridizedwithDNAprobes.AqerDNAextracGon,DNAtargetsaredetectedwithdifferentmethods(directhybridizaGon,PCR,realGmePCR).

Classifica5onofNucleicacidprobetechniques

1)DNAprobe: ExtracGon+hybridizaGon(lowsensiGvity)2)PCR: ExtracGon+specificgeneamplificaGon+

hybridizaGon(noquanGficaGon)3)RT-PCR: RealTimePCR:specificityandquanGficaGonofDNA

extractedfrombacteria

Synthe5coligonucleo5desDNAsequences(primers/probes)

OurGenomicTarget:rRNA16ssequences

Theribosomeiscomposedoftwosubunits.The30SsubunitisthesiteoftranslaGoniniGaGon.16Shasgotuniversal,consensusandspecificsequencesOneofthemostimportantDNAsequencesdatabaseforbacteria

hvp://www.hybridmedicalanimaGon.com

16SDNAcodingforrRNA16scontainhighlyconservedregionsandextremeheterogeneouspartsthatmakeitidealfordisGncGonbetweenspecies.

rRNA16SsequencesB:rRNA16s

Real Time PCR method DNA from each pathogen are amplified. The control of the cycle number allow to quantify initial bacterial number.

100 000 fg 10 000 fg 1000 fg 100 fg 10 fg

Fluo

resc

ence

inte

nsity

Ct

RealTimePCRwith16SNucleicacidØ  easycollecGonandtransport(nosurvival

ofthebacterianecessary)Ø  fastanalysisvscultures(severalhours)Ø  highsensiGvity(LOD103)beverthan

culturemethodsØ  highspecificityØ  OKnonculGvablespeciesØ  LimitedmutaGonof16Sanaerobicstrain

NoevaluaGonofanGbioGcsensiGvityNoevaluaGonofvirulence

Evalua&onofan&bio&cresistancecouldbedonewithspecificprobes

Institut Clinident test : Perio-Analysis Real Time PCR for Peri-implantitis prevention

•  DNAExtracGonfromthepaperpoints•  DNAamplificaGonwithDNAprobesandprimers

–  QuanGficaGonpermicrobialtarget–  TotalOralCount %foreachtarget/TotalOralCount

Perio-analyse®isaRealTimePCRtestbasedonmicrobialDNAextrac5onandMulGparametricquanGficaGonofDNAbacterialspecificsequencesfromGingivalCrevicularFluidorPerio-implantSulcusFluid.

Theproposedtestismeasuring9bacteria+CandidaAlbicans+TotalOralCount:§Aggrega&bacterac&nomycet-emcomitans§Porphyrom-onasgingi-valis§Tannerellaforsythensis§Treponemaden&cola§Prevotellaintermedia§Peptostreptococcusmicros§Fusobacteriumnucleatum§  Campylobacterrectus§  Eikenellacorrodens§  CandidaAlbicans+TotalOralBacteriaCount

A service provided by Institut Clinident

1.prélèvement 2.insertion dans tube 3.formulaire

4. envoi postal

Prescription form and instructions

Method

DNA extraction from paper point

PCR ADNr 16S

Quantification by Real time PCR

•  MucosiGs≈GingiviGs•  Peri-implanGGs≈Periodontaldisease

Samebacteria&microflora(Mombelli2002,Quirynen2002,Renvert2006,Mavout2009,Pye2009,Heydenrijk2002)

SocranskyclassificaGon

*Tannerellaforsythia=previouslyTannerellaforsythensis**Parvimonasmicra=previouslyPeptostreptococcusmicros

TannerellaForsythia(Tf)*

Parvimonas Micra (Pm)**

Increaseofthe

patho

genicit,

Microflora to be tested

Foundinto7.1%to19.6%ofpaGenthavingchronicperiodontaldisease(Sardietal,ArchOralBiol2011;56(10):1098-105.

•  AacouldbealonewithhighquanGtyinagressiveperiodontaldisease•  Aacannotbetreatedonlymechanically(intra-cellularbacteria)•  Boneand5ssuedestruc5oneveninlowquan5ty•  SpecificanGbioGctreatment(Tetracycline)associatedwithmechanicaltreatment

(newquinoloneisalsoefficient).Metronidazoleisnotefficient.

•  OsteoplastycouldberecommendedfortotaleliminaGon(intra-cellular)•  Synthesisofendotoxinsabletodamageleucocytes&monocytes•  Presentinendocarditesandpneumonia

PorphyromonasgingivalisBone destruction . Destruction of the immunoglobulins and collagen tissue Active in limited quantity (105 CFU par sample)

TannerellaForsythia(Tf)*

Limited action if low quatity Destruction of proteins (proteolytic activities)

Treponemaden3cola

Eliminated by a mecanical treatment Destruction of proteins (proteolytic activities) Need to ba associated with other pathogens (red complex, orange complex…) to grow in vivo

Tannerellaforsythia RedcomplexAggressivebacteria

Prevotellaintermedia

Very well known resistance of Pi to Ampicillin Most of P. intermedia strains are know to be resistant to titanium bactericide effect (implant material or surface treatment)

ParvimonasmicraSynergies with other bacteria for bone and tissue destruction .

Always present . High quantity if GUN and Peri-implantitis Associated with stress

Parvimonas Micra (Pm)**

Fusobacteriumnucleatum

Virulent factor are not know

Campylobacterrectus

Commensal bacteria Cytotoxic effect on epithelial cells (high concentration)

Eikenellacorrodens

Synergywithotherbacteria

•  C.albicansiscommensalandaconsGtuentofthenormalfloracomprisingmicroorganismsthatliveinthehumanmouth

•  Candidaalbicanscancolonizesulcusandberesponsibleforperiodontaldisease•  CandidaalbicanscannotbetreatedwithanGbioGcs(notabacteria)•  C.albicansiscapableofcolonizingtheperiodontalpocketsinpaGentswithchronic

periodonGGsandpaGentswithaggressiveperiodonGGs.•  AnGbioGctreatmentcouldfacilitedevelopmentofCandidaalbicans•  LocalanGfungalscouldberecommendedforeliminaGon•  PrecisequanGficaGonofCandidaalbicansisnotpossible

Complex PathogensPeriodon55s Chronic

gingivi-5s

Chronic RapidlyProgres-sive

Juvenile Pre-pubertal

Peri-coronary

Necro5zingulcera5ve

Aggrega5bacterac5nomycetemcomitans(Aa)

RedComplex

Porphyromonasgingivalis(Pg)

Tannerellaforsythensis(Tf)

Treponemaden5cola(Td)

OrangeComplex

Prevotellaintermedia(Pi)

Peptostreptococcusmicros(Pm)

Fusobacteriumnucleatum(Fn)

Orangecomplexassociated

Campylobacterrectus(Cr)

Greencomplex

Eikenellacorrodens(Ec)

CandidaAlbicans(Ca)

BasedontheSocransky’sdefini5on

Pathogenicity

NEW

Pathogenicity

Result presentation

Microbiologicalresults<Numericdata> <Graphicdata> +

Bacteria Pathogenicload

Pathogenic threshold Status

Total Count 3,6E+09Aggregatibacter actinomycetemcomitans 7,6E+05 1,0E+03 +++

Porphyromonas gingivalis 5,8E+07 1,0E+05 +++Tannerella forsythensis 1,1E+05 1,0E+05 ++Treponema denticola 0,0E+00 1,0E+05 +Prevotella intermedia 1,4E+06 1,0E+05 +++

Peptostreptococcus micros 1,0E+06 1,0E+06 ++Fusobacterium nucleatum 1,7E+07 1,0E+07 ++

Campylobacter rectus 1,8E+05 1,0E+06 +Eikenella corrodens 4,1E+06 1,0E+07 -Candida albicans 1,9E+08

Periodontalpocketflora NotbalancedSymptom Aggressive/activeperiodontaldisease

Pathogenreduction Withaprotocoltodisruptbiofilm(surgicalplastysuggested)Homecare Withlocalantimicrobials

Doxycycline+MetronidazoleReduceCandidaalbicanswithoralantifungaltherapy

Reassessment Comparepre-andpost-treatment(12weekspostantibiotictreatment)

Implantplacement NotrecommendedVeryhighriskforPeri-implantsHighriskforrapidbonelosswithouttreatment

*SuggestedantibiotherapyoptionisatClinician'sdirection.(subjecttoallergy,druginteraction,medicalconsideration)

Possiblecomplications

Clinicalinterpretation

Therapeuticrecommendation

Reassessmentrecommendation

Riskassessment

Antibiotictherapy

Type4-6 Type5

1

10

100

1000

10000

100000

1000000

10000000

100000000

1E+09

1E+10

Aa Pg Tf Td Pi Pm Fn Cr Ec Ca

• Stepbystep• Iden5fica5on• Quan5fica5on

• Clearerimagewithbacteriagroupcolorcoding• Easiertounderstand/interpret• Supportfortreatmentdecisions

PaGentDiagnosis(Saliva)

TreatmentdecisionAnGbioGcchoice

Monitoring(3monthsand12months)

TissuecontrolbeforeplacingtheImplant(T-14days)

PreventPeri-implanGGsandimplantfailure(3monthsand12months)

AnnualMonitoringcontrol

When?

Rational treatment plan

Firstvisit

PeriodontalDiagnostic

Treatment

TreatmentEvaluation

Followup Surgery

Implantinstallation Evaluation

Evaluation

Peri-implantitis treatment

Followup

Perio-Analyse:SoproCare:

Perio-AnalyseSaliva

Sulcus

Testedmatrices Results Recommenda&ons

saliva presenceofpathogenicbacteriawithoutAaand/orPg

-controlalldentalpocket,depth,radicularsurfacing -sub-gingivalscaling -hygieneprotocol

saliva presenceofAaand/orPg -controlalldentalpocketdepths,andusePerio-Analyseforeachdentalpocketwithmorethan4mm -followdentalpocketPerio-AnalyserecommendaGonswiththenewresults

dentalpocket presenceofpathogenicbacteriaunderthethreshold

-radicularsurfacing -sub-gingivalscaling -hygieneprotocol -noneedforanGbioGcs

dentalpocket presenceofAaoverthethresholdassociatedornotwithotherpathogenicbacteriaoverthethreshold

-radicularsurfacing -sub-gingivalscaling -hygieneprotocol -possiblebonesurgerywith/withoutflap -tetracyclinecouldberecommended

ThepossiblesituaGonsarepresentedschemaGcallywithinmicrobiologicalcomplexespresenceorabsenceintosalivaorpocket.

• Allmedicalactsarecondi5onedbyitsmedicalvalueandeconomicalsustainability

Economicalvalue Medicalvalue

- Peri-implanGGsriskevaluaGon ++++ +++- TreatmentdecisionforPeri-implanGGsdisease +++ +++- Treatmentdecisionforperiodontaldisease - ++- PeriodontalriskevaluaGon + ++

- RightGmetoplacetheimplant - +++- Implantsurfacestrategy ++ ++++- tesGngatannualvisit +++ ++++- Medicolegalresponsibility ++++ ++- Medicalinsurances +++ +

Medical and Economical value

Rational of Periimplantitis Diagnostic

BenefitsperceivedbyPeriodon5st

•  Cleardiagnosis/infecGouscauseevaluaGonbeforetreatment/surgeryandanGbioGcdecision

•  NewtoolsforclearcommunicaGonwithpaGents•  realmonitoringoftheperiodontalsituaGon•  treatmentefficiency•  biologicaljusGficaGonforlongtreatmentperiod

•  ReducGonoftheriskfortreatmentfailureorsecondinfecGon

•  MoGvaGonofthepaGent

Benefitsperceivedbyden5stsusingimplants

•  RiskevaluaGonbeforeplacingtheimplant•  LimitresponsibilityincaseofimplantfailureifthepaGentwouldliketoproceedwithimplantplacementwithoutefficienttreatmentorina“poorcondiGon”

•  ReducGonofrisk&earlydiagnosistopreventperi-implanGGs

•  MoGvaGonofthepaGentforbeverhygieneandimplantcleaningprotocol(reducebacteria=saveimplant)

Benefitsperceivedbypa5ent

•  TheywillunderstandthepathologycausedbybacterialinfecGonandwillimprovedentalmaintenance

•  MoGvaGonofthepaGent•  Theywillpreserveinvestmentandreduce“possible“infecGousrisk

•  GoodappreciaGonfortheknowledgeofthedenGst

ECONOMICALVALUEFORTHEDENTISTS

ECONOMICALVALUEFORTHEPATIENT

PREVENTPERI-IMPLANTITIS SAVEATOOTHANDBONE

MEDICALVALUEFORTHEDENTISTS

AnnualMonitoringthesurfaceoftheperi-implanttoreducefinancialriskofimplantfailureduringanacceptableperiodofGme

BeverDiagnosis=Bevertreatment

Fasterthetreatment

DonotcompromisethechanceforsuccessfulboneregeneraGon

Monitoringthesurfaceoftheperi-implantbever

protecGonofmedicolegalresponsibilityforthedenGsts(&couldsave

money)

Values for the dentist and patient

PersonalizedtreatmentHighersuccessofmypracGce(Perio&implantology)ExternalopinionviatheanalyGcalreportBiologicalsupportHightechposiGonofmypracGceandbevervisibilityofmyClinicSecuretheprognosisforPeriodontaltreatmentReducePeri-implanGGsrisk–improvecuraGvetreatment

Whatistheinterestformypa5ent?

WhatismyinterestasaDen5st?

FAQ

WhynotonlycollecGngthesaliva?- becausethepresenceofperiodontalbacteriaintothesalivaisariskfactor,butnotadiagnosGc.OnlypresenceofnonequilibredmicroflorainthesulcuscouldbeconsideredasadiagnosGctoolandshouldbetreated.

- HowcanIusePerio-analyseinmypraGce- FornewpaGent- PaGentwithperiodontaldiseasetobetreated- PaGentatrisk,goingtohavedentalimplantaqerperiodontaldisease- PaGentatriskhavingdentalimplant(annualcontrol)- PaGentwithdentalimplanthavingmucosiGsorperi-implanGGs- PaGentatgeneGcrisk(InterleukinmutaGon)- DiabeGcpaGent- PregnantpaGenthavingperiodontaldiseaserisk

- Whatbacteriaareathightriskforrapidbonelost?- AaandPg

- WhyAbisassociatedwithsurgery/mecanicaltreatment- Aaisintracellularbacteria.CertainbacterialikeAaandPgarenotstrictanaerobicandcouldsurviveinpresenceofoxygenaqersurgery

FAQ

WhyCandidaalbicanscouldbeatrisk?- becauseinpresenceofanGbioGcandaqerbacteriareducGonandlimitedcompeGGon,Cacangrowandfullycolonizethesulcus.

- Whataretheunitsusedforbacteriaload- 10000=104=E+04=10000bacteriainthetestedsulcus

- WhatisPCR- PCRisamplificaGonofspecificzoneofDNA- Eachamplifiedzoneisspecificfromabacteriaspecies

- HowlongisthePCRprocess- DNAextracGon2hours- PCRpreparaGon1hour- PCRrunGme2hour(about10paGentsperrun)

- WhenshouldItestforthesecondGme- 3monthsaqerthefirstsamplingandanGbioGcuse

- AnGbioGcrecommendaGon- Aaaloneorwithredcomplex=Tetracycline- Redcomplex>thethreshold=Penicillin- Redcomplex+Pi>thethresholdorPialone=Metronidazole

FAQ

- MayIuseprobioGc?- yesaqerPeriotreatment,probioGcwillassureamicrobialcompeGGonandreducetheriskfornewinfecGon.ProbioGcsarenoteffecGveasacuraGvesoluGon.

-HowmanypaperpointshouldIused?- minimum2persite.10paperpointsforapoolsample

- HowlongisstableDNAonpaperpointatroomtemperature- >2weeks

- HowmanysamplescanberunperdaybyClinident- about150/day- Labisclosed2weeksperyear(1weekinAugustand1weekendofDecember

- WhataretheDNAtargetandDNAstandardofthePCRdevelopedbyClinident- Ribosomal16SDNAsequences- QualibratedDNAfromDSMZ(Germany)andInsGtutPasteur(France)

FAQ

AixenProvenceintheSouthofFrance

Loca5onofthelaboratory

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