Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and...

Methods of detection ofActinobacillusactinomycetemcomitans,Porphyromonas gingivalis andTannerella forsythensis inperiodontal microbiology,with special emphasis onadvanced molecular techniques:a reviewSanz M, Lau L, Herrera D, Morillo JM, Silva A: Methods of detection ofActinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerellaforsythensis in periodontal microbiology, with special emphasis on advancedmolecular techniques: a review. J Clin Periodontol 2004; 31: 1034–1047. doi:10.1111/j.1600-051X.2004.00609.x. r Blackwell Munksgaard, 2004.

AbstractBackground: Certain specific bacterial species from the subgingival biofilm havedemonstrated aetiological relevance in the initiation and progression of periodontitis.Among all the bacteria studied, three have shown the highest association withdestructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa),Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, therelevance of having accurate microbiological diagnostic techniques for theiridentification and quantification is clearly justified.

Aim: To evaluate critically all scientific information on the currently availablemicrobial diagnostic techniques aimed for the identification and quantification of Aa,Pg and Tf.

Summary: Bacterial culturing has been the reference diagnostic technique for manyyears and, in fact, most of our current knowledge on periodontal microbiology derivesfrom cultural data. However, the advent of new microbial diagnostics, mostly based onimmune and molecular technologies, has not only highlighted some of theshortcomings of cultural techniques but has also allowed their introduction as easy andavailable adjunct diagnostic tools to be used in clinical research and practice. Thesetechnologies, mostly polymerase chain reaction (PCR), represent a field of continuousdevelopment; however, we still lack the ideal diagnostic to study the subgingivalmicroflora. Qualitative PCR is still hampered by the limited information provided.Quantitative PCR is still in development; however, the promising early results

Mariano Sanz1,2, Laura Lau1,David Herrera1,2, Juan ManuelMorillo1 and Augusto Silva3

1Laboratory of Microbiology; 2Section of

Graduate Periodontology, Faculty of

Odontology, University Complutense;3Center for Molecular Biology, National

Center for Scientific Research, Madrid, Spain

J Clin Periodontol 2004; 31: 1034–1047 doi: 10.1111/j.1600-051X.2004.00609.x Copyright r Blackwell Munksgaard 2004

reported are still hampered by the high cost and the equipment necessary for theprocessing.

Conclusion: Quantitative PCR technology may have a major role in the near future asan adjunctive diagnostic tool in both epidemiological and clinical studies inperiodontology. However, culture techniques still hold some inherent capabilities,which makes this diagnostic tool the current reference standard in periodontalmicrobiology.

Key words: Actinobacillusactinomycetemcomitans; culture; diagnosticmethods; microbiology; Porphyromonasgingivalis; polymerase chain reaction;periodontitis; Tannerella forsythensis

Accepted for publication 8 March 2004

Destructive periodontal diseases arechronic inflammatory conditions char-acterised by connective tissue andalveolar bone destruction, eventuallyleading to tooth loss. A substantialnumber of publications have reportedthat certain microorganisms from thesubgingival microbiota, particularlyGram-negative anaerobes, are the majoraetiological factors of chronic andaggressive periodontitis (Dzink et al.1985, Slots 1986, Newman 1990).Although the subgingival microenviron-ment in the periodontal pocket ischaracterised by a wide diversity, withover 300 species having been isolatedfrom different individuals and as manyas 40 from a single site, only a fewspecies have been associated with dis-ease (Moore & Moore 1994). Despitethe difficulty in identifying all themembers of the oral microbiota andunderstanding how they interact witheach other and with the host, a limitednumber have demonstrated a clearaetiological role and these have beenidentified as periodontal pathogens(Genco 1996). Evidence for aetiologyis based on the fulfilment of severalcriteria described by Socransky (1970).Using these criteria, strong evidence hasbeen demonstrated for Actinobacillusactinomycetemcomitans (Aa), Porphyr-omonas gingivalis (Pg) and Tannerellaforsythensis (Tf) (formerly Bacteroidesforsythus), as concluded at the WorldWorkshop in 1996 (Genco 1996).

Aa is a member from the genusActinobacillus, belonging to the familyof Pasterurellaceae. It is a Gram-negative, small rod (1–1.5–0.5mm),capnophilic facultative anaerobe. Inculture, colonies grow in small flat,circular colonies that have slightlyblurred borders and a translucent out-look in solid media, sometimes with atypical inner star-like morphology(Zambon 1985, Alsina et al. 2001)(Fig. 1). Several lines of evidencesupport its aetiological role as a trueperiodontal pathogen, mostly in relationto aggressive periodontitis (Fives-Tay-lor et al. 1999, Slots & Ting 1999,

Socransky et al. 1999). This bacterialspecies has a wide intra-specific diver-sity defined by its six serotypes (Hen-derson et al. 2002). In the oral cavity,serotypes a and b are the most frequentin Caucasians, serotype b being mostfrequently associated with localisedaggressive periodontitis and therefore,the most virulent and pathogenic (Zam-bon 1985, Zambon et al. 1988, Albandaret al. 1991, Preus et al. 1994, Haraszthyet al. 2000a, b, Henderson et al. 2002).

Pg belongs to the genera Porphyr-omonas from the family Bacteroida-ceae. These bacteria are Gram-negativestrict anaerobic coco-bacilli. They growin culture media forming convex,smooth glossy colonies of 1–2-mmdiameter, which demonstrate a progres-sive darkening in the centre, because ofthe production of protoheme, the sub-stance responsible for the typical colourof these colonies (Fig. 2) (White &Mayrand 1981, Sha & Collins 1988,Ohta et al. 1991, Haraszthy et al.2000b). Several lines of evidence sup-port its aetiological role as a trueperiodontal pathogen, more likely asso-ciated with chronic periodontitis (Holt etal. 1999, Slots & Ting 1999, Socranskyet al. 1999). Its importance as aperiodontal pathogen is also highlightedby the research efforts aimed at devel-oping a vaccine aimed at immunisationagainst this bacterial species and thusprevent chronic periodontitis (Gibson &Genco 2001, Nakagawa et al. 2001,Rajapakse et al. 2002, Yang et al. 2002).

Tf is a non-pigmenting saccharolyticanaerobic Gram-negative rod. For along time, it was denominated Bacter-oides fusiformes. It was then renamedBacteroides forsythus by Tanner et al.(1986), and very recently a new namehas been proposed and accepted, Tf(Sakamoto et al. 2002). Its habitat is thegingival sulcus, and it is usually isolatedin periodontal pockets, although itspresence has also been detected in thetonsils, dorsum of the tongue and saliva.This bacterium was not considered atrue periodontal pathogen until recently,mostly because of its fastidious growth

in culture media. These difficulties inculturing are related to the lack ofcapacity of Tf to synthesise N-acetyl-muramic acid (NAM), an essentialcomponent of cell wall peptidoglycan.Therefore, it grows poorly in pureculture, unless the medium is supple-mented with NAM, or by growingtogether with other microorganisms(Fig. 3).

Moderately strong evidence has beendemonstrated for other bacteria isolatedfrom the subgingival microbiota such asCampylobacter rectus (Cr), Eubacter-ium nodatum, Fusobacterium nucleatum(Fn), Peptostreptococcus micros (Pm),Prevotella intermedia (Pi) and P.nigrescens (Pn), Streptococcus interme-dius and various spirochetes, such asTreponema denticola (Td) (Genco1996). Most of these pathogens are

Fig. 1. Colonies of Actinobacillus actino-mycetemcomitans growing on Dentaid-1medium.

Fig. 2. Colony of Porphyromonas gingiva-lis, close to another colony of Prevotellaintermedia/nigrescens, growing on bloodagar medium, supplemented with haeminand menadion.

Detection of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis 1035

members of the resident oral microbiotaand are usually present in healthy orgingivitis subjects. Therefore, theiraetiologic role is less evident. Prelimin-ary evidence of periodontal pathogeni-city has been considered for Eikenellacorrodens (Ec), enteric rods, Pseudo-monas species, Staphylococcus speciesand yeasts (Genco 1996). Some of thesespecies, such as Pseudomonas speciesor enteric rods species, are rarely found,and in relatively low numbers, inpopulations from Western countries;therefore, their aetiologic role alsoremains unclear.

More recently, viruses includingcytomegaloviruses, Epstein–Barr virus,papillomavirus and herpes simplex virushave been proposed to play a role incausing periodontal diseases (for areview, see Contreras & Slots 2000,Slots & Contreras 2000). The presenceof these viral genomes has been isolatedfrom periodontitis lesions in cross-sectional studies. These genomes havebeen found in chronic periodontitis(Contreras & Slots 1996), aggressiveperiodontitis (Michalowicz et al. 2000)and periodontitis associated with sys-temic diseased patients (Contreras et al.2001). These authors hypothesise thatenvironmental or systemic factorswould allow for the activation of theseviruses, whose inhibitory impact onvarious host factors would allow forthe accumulation of periodontal patho-gens and development of destructiveinflammatory disease.

Even though the aetiological role ofAa, Pg and Tf in periodontitis seemsuncontentious, the use and utility ofdiagnostic tests aimed at identifying andquantifying the presence of these bac-teria in periodontitits patients remainvery controversial. The purpose of thisreview is, firstly, to analyse criticallythe available evidence on the rationaleof the use of microbial diagnosis in themanagement of periodontitis patients,and secondly, to review the differentdiagnostic methodologies used to iden-

tify and quantify these putative perio-dontal pathogens, with special emphasison those methods aimed at clinical useand based on molecular technologies.

Rationale for microbial diagnosis inthe management of patients withperiodontitis

For microbial diagnosis to be of value, itneeds to have an impact on the overalldiagnosis and/or treatment planning,resulting in a superior treatment out-come, and/or providing a clear benefit tothe patient.

The utility of microbiological testingfor diagnosing the different forms ofdestructive periodontal diseases remainscontroversial. The limitations of testingfor the presence or absence of Pg andAa, aimed at distinguishing subjectswith aggressive periodontitis from sub-jects with chronic periodontitis, wasclearly shown in the systematic reviewrecently published by Mombelli et al.(2002). Although the diagnosis ofaggressive periodontitis may be lesslikely in a subject with no detection ofAa, the sensitivity and specificity for apositive clinical diagnosis of aggressiveperiodontitis in the presence of thesebacteria are low and heterogeneous, andtherefore, the mere presence or absenceof these putative pathogens is not ableto discriminate subjects with aggressiveperiodontitis from those with chronicperiodontitis.

The utility of microbial identificationas an aid in the treatment planning ofpatients with periodontitis has beentested in a limited number of studies,mostly case reports dealing with aggres-sive or non-responding periodontitispatients (Levy et al. 1993, Rosenberget al. 1993, Renvert et al. 1996,Eickholz et al. 2001, Ishikawa et al.2002). The aim of most of these studieswas to guide in the selection ofadjunctive antimicrobial therapy basedon the microbial data. Only one of thesepublications reported a controlled study(Levy et al. 1993), where two differentgroups of periodontists developed theirtreatment plans based on the results ofadjunctive diagnostic microbiology(test), or just on standard clinicaldiagnosis (controls). The use of micro-bial diagnosis resulted in the use ofmore systemic antibiotics and lessperiodontal surgery. However, thelong-term outcome of these patientswas not reported. The rest of thepublications involved case reports,

where the use of microbial diagnosisguided periodontal therapy. In most ofthese studies, the patients improvedfollowing treatment, some even show-ing dramatic improvements. The lack ofappropriate controls, however, makesthe interpretation of these results diffi-cult and therefore, the utility of micro-bial testing in developing specifictreatment plans cannot be ascertained.

The utility of microbiological testingas an indicator of healing or diseaseprogression has been suggested byseveral prospective studies, where thedetection or lack of detection of putativeperiodontal pathogens was significantlyassociated with a different clinicalresponse (Wennstrom et al. 1987, Haf-fajee et al. 1991, 1995, 1996, Ramset al. 1996, Dahlen & Rosling 1998,Buchmann et al. 2000, Chaves et al.2000, Tran et al. 2001). In most of thesestudies, the absence of these pathogenswas a better predictor of periodontalhealth than their presence as a predictorof periodontal disease (Wennstrom et al.1987, Dahlen & Rosling 1998). Somestudies showed that the presence ofthese pathogens above certain criticallevels increased the risk for perio-dontitis recurrence. Rams et al. (1996)showed a 2.5 times increased risk ofdisease recurrence when Aa, Pg, Pi, Cror Pm were detected at baseline.Similarly, Haffajee et al. (1995, 1996)showed a significant increased risk ofattachment loss when Aa was present atthreshold levels above 104, and Pgabove 105. Chaves et al. (2000) showedthat the presence of Pg was predictiveof disease progression and bone loss,and Tran et al. (2001) demonstrated thatpatients with persistent Tf were 5.3times more likely to develop loss ofattachment than those without Tf.Although these prospective studiesmonitoring patients after therapy wouldindicate that the use of microbial testingcould aid in the selection of a moretargeted therapy, mostly in patients withaggressive or recurrent periodontitis, thelack of clinical trials with adequatecontrols prevents from demonstratingthe real value of microbial diagnosis.Therefore, the available evidence doesnot fully prove the utility of microbio-logical testing in periodontitis patients.

Microbiological diagnostic methods

Different methods have been used forthe detection of putative periodontalpathogens in subgingival samples. Some

Fig. 3. Colony of Tannerella forstythesis,growing on blood agar medium.

1036 Sanz et al.

of these methods have been strictlyused for research purposes, while othershave been adapted or modified for clini-cal use.

Bacterial culturing

Historically, culture methods have beenwidely used in studies aimed at char-acterising the composition of the sub-gingival microflora and are stillconsidered the reference method (goldstandard) when determining the utilityof a new microbial diagnostics inperiodontics. Generally, subgingivalplaque samples are cultivated anaerobi-cally and by using selective and non-selective media, together with severalbiochemical and physical tests, thedifferent putative pathogens can beidentified. The main advantage of thismethod is the possibility to obtainrelative and absolute counts of thecultured species. Moreover, it is theonly method able to characterise prop-erly new species and to assess theantibiotic susceptibility of the grownbacteria (Socransky et al. 1987, Green-stein 1988, Marchal et al. 1991, Lamsteret al. 1993). However, culture techni-ques have significant shortcomings.Culture methods can only grow viablebacteria; therefore, strict sampling andtransport conditions are essential. More-over, some of the putative pathogens,such as Treponema sp. and Tf, are veryfastidious and difficult to culture (Saka-moto et al. 2002). The sensitivity ofbacterial culturing can be rather low,especially for non-selective media, withdetection limits averaging 103–104 bac-terial cells, and therefore, low numbersof a specific pathogen in a subgingivalsample will be undetected. However,the most important drawback is thatculture requires specific laboratoryequipment and experienced personnel,besides being relatively time-consumingand expensive.

Methods based on immune diagnosis

Immunological assays use antibodiesthat recognise specific bacterial anti-gens, and the identification of thesespecific antigen–antibody reactionsallows the detection of target micro-organisms. This reaction can be visua-lised using a variety of techniques andreactions, including direct (DFA) andindirect (IFA) immunofluorescentmicroscopy assays, flow cytometry,enzyme-linked immunosorbent assay

(ELISA), membrane assays and latexagglutination (Greenstein 1988, Lam-ster et al. 1993). Both direct (DFA) andIFA are able to identify the selectedpathogen and quantify its percentage inthe flora by using a plaque smear. IFAhas been used mainly to detect Aa, Pgand Tf. Zambon et al. (1985) showedthat this technique was comparable withbacterial culture in its ability to identifyAa and Pg in subgingival plaquesamples. In fact, IFA demonstrated ahigher sensitivity when compared withculture, probably because of a lowerdetection limit. Comparative studiesindicated that the sensitivity of theseassays ranged from 82% to 100% fordetection of Aa and from 91% to 100%for detection of Pg, with specificityvalues of 88% to 92% and 87% to 89%,respectively (Zambon 1985, Zambonet al. 1985, 1986). Listgarten et al.(1995) compared the diagnostic utilityof IFA and culture for the detection ofAa. They showed a higher sensitivity ofIFA (41.8%) and a significantly higherdetection rate (39.4 % for culture versus81.8% for IFA). These authors alsocompared the diagnostic utility of IFAfor the detection of Pg and Tf whencompared with oligonucleotide probescomplementary to the hypervariableregion of the 16S rRNA of the targetbacteria. They also demonstrated sig-nificantly higher detection rates andhigher sensitivity than DNA probes forboth bacteria.

Cytofluorography or flow cytometryfor the rapid identification of oralbacteria involves labelling bacterialcells from a patient plaque sample withboth species-specific antibodies and asecond fluorescein-conjugated antibody.The suspension is then introduced intothe flow cytometer, which separates thebacterial cells into an almost single-cellsuspension by means of a laminar flowthrough a narrow tube (Kamiya et al.1994). The sophistication and costinvolved in this procedure precludes itswide usage

ELISA is similar in principle to otherradioimmunoassays, but instead of theradioisotope, an enzymatically derivedcolour reaction is substituted as thelabel. The intensity of the colourdepends on the concentration of theantigen and it is usually read photome-trically for optimal quantification. ELI-SA has been used primarily to detectserum antibodies to periodontal patho-gens; however, it has also been used inresearch studies to quantify specific

pathogens in subgingival samples usingspecific monoclonal antibodies. A mem-brane immunoassay has been adaptedfor chair-side clinical diagnostic use andhas been marketed (Evalusites, East-man Kodak, Rochester, NY, USA). Itinvolves linkage between the antigenand a membrane-bound antibody toform an immunocomplex that is laterrevealed through a colorimetric reac-tion. Evalusites has been designed todetect Aa, Pg and Pi (Boyer et al. 1996,Chaves et al. 2000). Snyder et al. (1996)found a detection limit of 105 for Aa and106 for Pg.

In summary, immunological assaysfor oral bacteria provide a quantitativeor semi-quantitative estimate of targetmicroorganisms. These methods haveshown a higher sensitivity and specifi-city than bacterial culturing for thedetection of target microorganisms(Aa, Pg and Tf); however, they requirethe use of monoclonal antibodies toassure high specificity and the detectionlimits are not significantly lower thanbacterial culturing (103–104). Thesetests also have the advantage of notrequiring stringent sampling and trans-port methodology to assure bacterialviability. However, they are limited tothe number of antibodies tested, they arenot amenable for studying antibioticsusceptibility and they lack the validityof well-controlled clinical studies.

Enzymatic methods of bacterial

identification

Tf, Pg, the small spirochete Td andCapnocytophaga species share a com-mon enzymatic profile, since they allhave a trypsin-like enzyme in common.The activity of this enzyme can bemeasured with the hydrolysis of thecolourless substrate N-benzoyl-DL-argi-nine-2-naphthylamide (BANA). A diag-nostic kit has been developed using thisreaction for the identification of thisbacterial profile in plaque samples(Perioscans, Oral B Laboratories, Bel-mont, CA, USA). Loesche (1992) pro-posed the use of this BANA reaction insubgingival plaque samples to detect thepresence of any of these periodontalpathogens and thus serve as a marker ofdisease activity. Using probing depthsas a measure of periodontal morbidity,they showed that shallow pockets exhib-ited only 10% positive BANA reac-tions, whereas deep pockets (7mm)exhibited 80–90% positive BANA reac-tions. These authors (Loesche et al.


1992) tested the diagnostic utility of theBANA test compared with culture, IFA,ELISA and DNA probes, renderingsimilar results with regard to sensitivityand accuracy (above 90% to detectcombination of these organisms). How-ever, a clear BANA reaction wasindicative of more than 105 BANA-positive organisms, which indicates arather high limit of detection. Beck et al.(1990) also used the BANA test as a riskindicator for periodontal attachmentloss; however, the obtained odds ratioswere not statistically significant. Insummary, this chair-side test, althoughvery easy and fast to use, has significantlimitations. Mainly, since it only detectsa combined and limited number ofpathogens, its negative result doesnot rule out the presence of otherimportant periodontal pathogens or eventhe tested pathogens when present inlow number.

The simplicity, quick response andeasy-to-read use made these enzymaticand immune-based diagnostic methodsideal for chair-side use. In the 1990sthey were commercially available(Evalusites and Perioscans). However,the lack of appropriate clinical trials tovalidate their diagnostic utility and theirintrinsic problems regarding low sensi-tivity (Evalusites) and low specificity(Perioscans) made them disappear fromthe market soon.

Molecular biology techniques

The development of techniques inmolecular biology, aimed at the detec-tion of bacterial pathogens, has allowednot only the acquisition of knowledge inmicrobial genetics but has also set thebases for the development of improveddiagnostic techniques (Holt & Pro-gulske 1988, Saiki et al. 1988, Gibbs1990). The principles of molecularbiology techniques reside in the analysisof DNA, RNA or the protein structureor function (Lewin 1993, Dawson et al.1996). The genetic material of a bacter-ium is composed of a chromosomalDNA and transferring, ribosomal andmessenger RNA. Chromosomal DNA isdispersed in the bacterial cell withoutany membrane envelope (Holt & Pro-gulske 1988). Diagnostic assays usingmolecular biology techniques requirespecific DNA fragments that recognisecomplementary-specific bacterial DNAsequences from target microorganisms.For the development of a microbiologi-cal diagnostic test using this technology,

it is therefore fundamental to be able toextract the bacterial DNA from theplaque sample and to be able to amplifythe specific DNA sequence of the targetperiodontal pathogens.

Different chemical, enzymatic orphysical methods have been used toobtain DNA of sufficient quantity andquality for its subsequent analysis bymeans of either DNA probes or the poly-merase chain reaction (PCR). Amongthem, organic chemicals or detergentshave been used to destroy the cellularcomponents (membranes and proteins)and thus avoid interference with thesubsequent DNA assays (Smith et al.1989a, b). As enzymes, lysozyme isused to cleave the bacterial wall andproteinase K is used to destroy theproteinic components of the cell (Leyset al. 1994). Physical methods such asheat allow disruption of the cell anddenaturation of the proteins. Subse-quently, the use of centrifugation andchromatographic columns enables theseparation and purification of DNA. Forperiodontal microbial diagnosis, most ofthe tests developed have used proteinaseK or boiling and centrifugation (Ting &Slots 1997, Umeda et al. 1998b). Oncethe DNA from the subgingival plaquesamples have been extracted and pur-ified, different diagnostic methods havebeen developed to specifically detectand in some cases quantify the targetperiodontal pathogens.

Nucleic acid probes

A probe is a known nucleic acidmolecule (DNA or RNA) from a speci-fic microorganism artificially synthe-sised and labelled for its detection whenplaced together with a plaque sample.DNA probes entail segments of a single-stranded nucleic acid, labelled withan enzyme or radioisotope that is ableto hybridise to their complementarynucleic acid sequence and thus detectthe presence of target microorganism.Hybridisation refers to the pairing ofcomplementary DNA strands to producea double-stranded nucleic acid. Thenucleotide base-pair relationship is sospecific that strands cannot annealunless the respective nucleotide strandsequences are complementary. Allhybridisation methods use radio-labelled or fluorescence-labelled DNAprobes that bind to the target DNA ofinterest, thus allowing its visualisation(Lawer et al. 1990, Loesche 1992,Nicholl 1994, Dawson et al. 1996,

Tanner et al. 1998, Crockett & Wittwer2001). DNA probes may target whole-genomic DNA or individual genes.Whole-genomic DNA is more likely tocross-react with non-target microorgan-isms because of the presence of homo-logous sequences between differentbacterial species. Currently, most ofthe probes used are oligonucleotidesranging from 20 to 30 nucleotides(Nicholl 1994, Dawson et al. 1996).

Whole-genomic probes for the detec-tion of Aa, Pg, Pi and Td have beendeveloped and tested, being the bases ofcommercially available diagnosticmethods (DMDxs, Omnigene, Cam-bridge, MA, USA). When comparedwith culture, van Steenberghe et al.(1999) reported a sensitivity of 96% andspecificity of 86% for Aa, and 60% and82%, respectively, for Pg in purelaboratory isolates. However, whentested in clinical specimens, both thesensitivity and specificity were reducedsignificantly, suggesting cross-reactivitywith unknown bacteria in subgingivalplaque samples. In order to overcomethis drawback, oligonucleotide probescomplementary to variable regions ofthe 16S rRNA bacterial genes have beendeveloped for the detection of variousperiodontal pathogens. These bacterial16S rRNA genes contain both regionsshared by different bacteria and shortstretches of variable regions shared onlyby specific organisms of the samespecies or genus (Moncla et al. 1990).When these oligonuclotide probes werecompared with culture in clinical sam-ples for the detection of Aa, Pg and Pi(Savitt et al. 1988), an effectiveness of100% in detecting Aa and Pi and of91% in detecting Pg was calculated atculture-positive levels (X103 cells).However, DNA probes were moresensitive than culture in detecting thesepathogens in samples from periodontitispatients (for example, Aa was detectedby probe analysis in 70% of localisedjuvenile periodontitis samples, but onlydetected in 10% by culture analysis).Conversely, when these probes werecompared with IFA for the detection ofPg and Tf (Listgarten et al. 1995), IFAshowed significantly higher detectionrates and higher sensitivity.

Checkerboard DNA–DNAhybridisation technology

Socransky et al. (1994) developed thistechnique by for the detection and levelsof 40 bacterial species commonly found

1038 Sanz et al.

in the oral cavity. The assay uses wholegenomic, digoxigenin-labelled DNAprobes and facilitates rapid processingof large numbers of plaque samples withrespect to a multiple hybridisation forup to 40 oral species in one single test.The DNA probes used in this technol-ogy are commonly be adjusted to permitdetection of 104 cells of each species,but can adjusted to detect 103 cells. Themethod requires sophisticated labora-tory equipment and expertise, and it ishighly specific. These factors have notled to generalisation of this assay fordiagnostic purposes. It is particularlyapplicable, however, for epidemiologi-cal research and ecological studies,since it does not require viable bacteriaand allows for the assessment of a largenumber of plaque samples and multi-tude of species (Haffajee et al. 1997,2001, Papapanou et al. 1997a, Levy et al.1999, Ximenez-Fyvie et al. 2000a, b,Feres et al. 2001, Haffajee & Socransky2001). Papapanou et al. (1997b) made acomparison of this method with culturefor the identification of subgingivalbacteria. The checkerboard technologyresulted in higher prevalence figures forhalf of the species tested (Pg, Pi, Pn, Fnand Tf) and statistically significanthigher bacterial counts for the majorityof the species. Both techniques rendereda reasonable degree of agreement.

PCR methodology

PCR has emerged as the most powerfultool for the amplification of genes andtheir RNA transcripts. This technique,developed in 1985, is the single techni-que used almost universally to studyDNA and RNA obtained from a varietyof tissue sources. PCR allows forobtaining high quantities of DNA in asimplified and automated fashion (Scho-chetman et al. 1988, Shibata 1992,Dawson et al. 1996, Jankowski & Polak1996). PCR typically begins with theisolation of DNA from a fresh tissuespecimen. By heating, the complemen-tary double strands of DNA split intosingle-stranded forms intended to act asthe template dictating the nucleotidesequence in vitro. Then, the amplifica-tion is followed using a DNA polymer-ase that requires a primer, or knownshort oligonucleotide sequence corre-sponding to the border of the region thatis amplified. For obtaining amplifiedfragments of constant length and in highquantities, a second primer, comple-mentary to the opposed chain, must be

used to anneal (bind) the template andflank the region of interest. This ampli-fication can be performed several times,known as cycles. In each cycle, theprocesses of complementary chainsdenaturation, primer hybridisation andprimer extension by means of thepolymerase take place (Fig. 4). Witheach cycle, there is an exponentialincrease in the quantity of DNA. Duringall these processes, the temperatureduring the cycle is critical in order tocontrol the double chain denaturationand the stability of the hybridisationbetween the model fragment and theprimer. In 1988, a thermo-stable DNApolymerase, isolated from the organismThermus aquaticus, known as Taqpolymerase, was developed (Saikiet al. 1988). This Taq polymerase hasallowed the automatisation of the reac-tion using specific appliances namedthermo-cyclers. This sequenced DNA isthen detected and visualised throughelectrophoresis in agarose gel andethidium bromure, obtaining a qualita-tive signal (Jankowski & Polak 1996,Neumaier et al. 1998).

Although PCR is an extremely sensi-tive technique, being able to detect evenone copy of the searched DNA fragment(Greenstein 1988), it has a number ofimportant limitations. Difficulties canbe encountered when studying smallquantities of DNA, since the ingredientsnecessary for PCR (oligonucleotideprimers, dNTPs, Taq polymerase) maybe exhausted before sufficient target isproduced. The specificity of the reaction

depends on many complex, interrelatedfactors, including oligonucleotide pri-mer size, annealing temperature andbuffer salt concentration. A majorlimitation of PCR is the susceptibilityof the process to contamination, parti-cularly in experiments intended todetect rare DNA sequences (Gibbs1990, Neumaier et al. 1998). In orderto overcome some of these drawbacks,different varieties of PCR have beendeveloped. Multiplex PCR allows theamplification of several target regions,placing all the necessary primers in onesingle reaction (Dangtuan & Rudney1996, Henegariu et al. 1997, Garcıaet al. 1998). Quantitative PCR allowsthe quantification of all DNA fragmentsdetected by PCR using specific controlsof known quantity (Doungudomdacha etal. 2001). Quantification of DNA byPCR has tremendous potential in perio-dontal microbial diagnosis since itallows the analysis of a large numberof samples relatively easily, providing ameasure of flexibility not permitted byother conventional laborious and time-consuming methods. In practice, how-ever, a number of technical challengeshad to be overcome to develop reliableand reproducibility quantitative PCR.One of the principal challenges in thistechnique is the nature of PCR productaccumulation. During the reaction, thereare two defined growth phases. At lowcycles, PCR product accumulates expo-nentially (exponential phase); however,at higher cycles, as template DNA andprimer are being consumed, the rate of

Fig. 4. Diagram depicting the polymerase chain reaction process defined by the three stagesthat comprise this technique: amplification is carried out by a DNA polymerase, once in eachcycle. Each cycle includes the denaturalisation or separation of complementary chains,hybridisation of primers with the original chains and the extension of the primer by thepolymerase. Between 30 and 40 cycles are necessary to obtain a significant amount of thestudied sequence.


product formation progressively decrea-ses until it ends (saturation phase). Toobtain reliable quantitative PCR, themeasurement of the obtained productmust be made during the exponentialphase of the reaction, since during thesaturated phase the results will beinaccurate. At present, the method ofchoice for quantitative PCR is contin-uous monitoring of the amount ofproduct at the end of each cycle. Thisis accomplished by real-time quantita-tive PCR. It uses the 50–30-endonucleaseactivity of Taq DNA polymerase todetect target sequences during PCR andincluded in the mixture is a shortfluorescent oligonucleotide probe as alabelling system. During PCR, the probehybridises to the target DNA, and Taqpolymerase cleaves the probe into short-er fragments, thereby releasing fluores-cence that is directly proportional to theamount of PCR product generated. Bymeasuring the amount of PCR producedat the end of each single cycle, PCRgrowth curves can be plotted andmeasurements can be taken from theexponentially expanding region of thereaction (Crockett & Wittwer 2001,Meuer et al. 2001).

PCR in the detection of periodontalpathogens

Since the advent of PCR technology,different microbiological tests havebeen developed for the detection ofAa, Pg and Tf using a variety of DNAextraction methods and primers (seeTables 1–3). Riggio et al. (1996)compared the detection of Aa and Pgin subgingival plaque samples in patientswith periodontitis using either PCR orculture. PCR was more accurate thanconventional culture methods for iden-tification of these periodontal pathogensin subgingival plaque samples anddemonstrated a higher frequency ofdetection of these target microorgan-isms. Ashimoto et al. (1996) developeda 16S rRNA-based PCR detectionmethod to determine the prevalence ofAa, Tf, Cr, Ec, Pg, Pi, Pn and Td.Matched results between PCR andculture occurred in 28% (Tf) and 71%(Aa) of the samples; the major discre-pancy occurred in the PCR-positive/culture-negative category. This is prob-ably because of the PCR lower detec-tion limit, ranging from 25 to 100 cells,in comparison with culture (104–105

cells). Eick & Pfister (2002) recentlycompared a commercial multiplex PCR

of 16S rDNA for Aa, Pg, Pi, Tf and Tdwith standard culturing. The PCR testwas able to detect Pg and Tf more oftenthan cultivation. Aa was detected insimilar numbers with both techniques.Most of the available PCR tests devel-oped to detect Aa, Pg or Tf have used, asprimers, the 16S rRNA genes (Umedaet al. 1998a, b, Takamatsu et al. 1999,Choi et al. 2000, Contreras et al. 2000,Darby et al. 2000, Mullally et al. 2000,Okada et al. 2000, 2001, Kamma et al.2001, Takeuchi et al. 2001, Tan et al.2001a, Avila-Campos & Velasquez-Melendez 2002). Only a small numberof PCR assays have used oligonucleo-tides derived from single copy genes asprimers, such as the Pg collagenase prtCgene or the fimbrillin fimA gene, the Aaleukotoxin lktA gene or the Tf proteaseprtH gene. 16S rRNA genes as primers,although very specific (cross-reactivityis rare), may not be appropriate forquantitative analysis since there are avariable number of these molecules percell, which prevents a reproduciblequantification. These PCR tests provideonly qualitative information (preva-lence) and therefore, their use fordiagnostic and prognostic purposes inclinical use is limited. Most of thesediagnostic tests have been used inecological studies assessing the preva-lence of the target microorganisms indifferent populations, both in health ordisease. Results from these studies arevery heterogeneous showing differentprevalences (ranging from 2% to 95%for Aa) of the target bacteria in differentpopulations both in health and disease(Tables 1–3). This heterogeneity mightindicate that the high sensitivity of thistechnique is able to detect low numbersof bacteria that might be irrelevantin terms of pathogenicity or the possi-bility of different serotypes fromthe same species being highly prevalentin some populations without leadingto pathogenicity.

The importance of a quantitativeassessment of the target bacteria hasled to the recent development of quan-titative PCR methods. The first assaysused end-point PCR (Fujise et al. 1995,Doungudomdacha et al. 2001). How-ever, this technique obtains PCR pro-duct in the saturated phase, disregardingthe early exponential phase; therefore,the amount of PCR product obtainedshows a weak correlation with the initialDNA quantity. To overcome this limita-tion, real-time PCR assays have beendeveloped. With this technology and by

using a single copy of these genes percell, a good correlation between thefluorescent signal measured and thenumber of cells was obtained (Lyonset al. 2000, Shelbourne et al. 2000).Morillo et al. (2003) tested a real-timePCR assay, based on single copy genesequence and on the SYBR Green Ichemistry, aimed at the quantification ofAa and Pg in subgingival plaquesamples. This assay demonstrated ahigh degree of specificity and a veryreproducible and consistent method toquantify these pathogenic species. Real-time PCR, although demonstrating ahigh degree of sensitivity, specificityand reproducible quantification,requires expensive laboratory equip-ment, which makes this method veryexpensive for routine diagnostic clinicalmicrobiology. Recently, Rudney et al.(2003) also reported a quantitative PCRassay for Aa, Pg and Tf. This proposedtechnique, although cheaper than real-time PCR, may be associated with ahigher variability, since it lacks detec-tion during the exponential phase.Moreover, the primers used are 16SrRNA genes, which makes a reproduci-ble quantification difficult.

This review clearly shows thatstandard PCR technology, althoughdemonstrating high sensitivity and spe-cificity for the identification of targetperiodontal pathogens, is unable toaccurately quantify them in clinicalsamples, and therefore its role as aroutine clinical diagnostic tool is lim-ited. However, the advent of quantita-tive PCR technology may circumventthis limitation and at the same time mayclearly improve some of the shortcom-ings of standard cultural techniques.These quantitative PCR assays must bevalidated in clinical studies in order todemonstrate their diagnostic utility,and actual cost–benefit evaluation oftheir use in routine clinical diagnosismust also be carried out since theyrequire expensive and sophisticatedtechnology. Moreover, it is importantto keep in mind that although quanti-tative PCR technology may have amajor role as an adjunctive diagnostictool in both epidemiological and clini-cal studies in periodontology, culturetechniques still hold some inherentcapabilities, such as its ability todetect multiple bacterial species coin-cidentally, to detect unexpected bac-teria or to allow the determinationof antibiotic resistance, which stillmakes this diagnostic tool the current

1040 Sanz et al.

Table1.Prevalence

ofActinobacillusactinomycetem

comitansusingPCR

technique

Reference

Studygroup

Sam

plingmethod

DNA

extraction

Primers

Prevalence

Yuan

etal.(2002)

328subjects,7–12years

old,Taiwan

Curetteanddilutionin

200ml

distilled

water

Dilutionin

250ml

Tris-EDTA/Triton

X-100;boiling,10min;centrifuge,

10,000�

g,3min;store

supernatant

lktA

gene

7years

old,1.3%;8years

old,

3.5%;9years

old,2.8%;

10years

old,8.3%;11years

old,

7.7%;12years

old,9.5%

Yuan

etal.(2001)

246periodontalsubjects:

105non-insulin-dependentdiabetes,

141nodiabetes,Taiwan

Curetteand200ml

Tris-EDTA/TritonX-100

Boiling,10min;centrifuge,

10,000r.p.m

.,3min;store

supernatant

ktA

Prevalence:5.7%

sites,

nodifference

betweengroups

Okadaet

al.(2000)

104subjects,12years

old:

21periodontitis,73gingivitis,

10healthy,Japan

Dentalbrush

anddilutionin

distilled

water

Centrifuge,

1600�

g,20min;chem

ical

purificationofpellet

16SrRNA

Total,7.7%;healthy,4.8%;

gingivitis,6.8%;periodontitis,

20%

Takam

atsu

etal.(1999)

26patients:11adultperiodontitis,

15EOP,Japan

Paper

pointanddilutionin

100ml

distilled

water

Boiling,10min

16SrRNA

Before

treatm

ent,30.8%;post-

treatm

ent,19.2%

Tan

etal.(2001b)

92adultsubjects,25–65years

old:

50healthy,15moderateperiodontitis,

27severeperiodontitis,China


500ml

reducedtransportmedium

RTF

Centrifugeanddilutionofpelletin

200ml

RTF,withsm

allglass

beads;incubation,

941C,10min;cooling,5min

ktA

Total,74%;periodontitis,69%;

healthy,78%

Choiet

al.(2000)

29severeperiodontitis,20healthy,

Korea

Paper

pointand1mlRTF

Dilutionin

100ml

andcentrifuge13,000r.p.m

.,10min;dilutionin

Tris-EDTA/SDS

Consensus

bacterial

16S-rRNA

andhybridization

withspecificoligos

Periodontitis,89.7%;healthy,5%

Tan

etal.(2002)

146patients:113chronic

periodontitis,

33aggressiveperiodontitis,Singapore


500ml

RTF


200ml

RTFandglass

beads;incubation,941C,

10min

txA

cdt,ABC

xtA:Ch.P.,67%;Ag.P.,91%;

genotypecdt:Ch.P.,4%;Ag.P,

33%

Riggio

etal.(1996)

43patients:United

Kingdom


500ml

RTF

Mix

90ml

sample

and10ml

Tris-EDTA/Triton

X-100;boiling,5min

lktA

Prevalence,40%

Doungudomdachaet

al.

(2001)

50adultperiodontitis,United

Kingdom

Curetteand700ml

RTF


250ml

distilled

water

lktA

Diseasedsites,91.8%

Darbyet

al.(2000)

57periodontitis:33severeperiodontitis,

24generalized

EOP,United

Kingdom


500ml

Tris-EDTA

Mix

withTris-EDTA/TritonX-100;

boiling,5min

lktA

Severeperiodontitis,3%;EOP,

20.8%

Mullally

etal.(2000)

42EOP:25localized,17generalized,

Ireland


500ml

PBS

Centrifuge,

14,000r.p.m

.,5min;

dilutionofpelletin

500ml

PBS

twomore

times

16SrRNA

Prevalence,19%;28%

localized

periodontitis;5.9%

generalized

periodontitis

Kam

maet

al.(2001)

16post-treatment(m

echanical

and

antimicrobialtherapy)patients

insupportivephase,

Greece

Paper

point

Dilutionin

500ml

Tris-EDTA

anddisperse

invortex.Mix

250ml

suspensionand250ml

water;centrifugeandresuspendpellettwo

more

times;boiling,10min;centrifuge

andstore

supernatant

16SrRNA

Prevalence:activesites,18.8%;

non-activesites,6.3%

Umedaet

al.(1998a)

199subjects:52Caucasian,

49Afroam

erican,48Asian,

50Hispanic,USA

Paper

point

Dilutionin

500ml

distilled

water

anddisperse

invortex;centrifugeandresuspensionof

pellettwice;

store

supernatant

16SrRNA

Prevalences:Caucasian,

Afroam

erican,Asian,Hispanic

healthy/ging:23.1%,50%,29.4%,

50%

initialperio.:7.7%,27.8%,

36.4%,50%;mod.perio.:8.3%,

21.4%,75%,56.3%;sev.perio.:

35.7%,30.8%,50%,58.3%

Contreras

etal.(2000)

218patients:EOP,USA

Paper

point

Dilutionin

500ml

Tris/Tween-20/proteinaseK;

incubation,601C,1h;incubation,971C,15min;

boiling,10min;centrifugeandstore

supernatant

16SrRNA

Caucasian,36%;Hispanic,49%;

Asian,26%;Afroam

erican,38%

Avila-Cam

pos&

Velasquez-M

elendez

(2002)

100subjects:50periodontitis,

50healthy,Brazil

Paper

pointanddilutionin

VMGA

IIImedium

Mix

sample

with500ml

water;centrifugeand

resuspendpellettwice;

boiling,10min

16SrRNA

Prevalence:healthy,70%;

periodontitis,90%

Fujise

etal.(2002)

104subjects,1149sites,Japan

Paper

point

Dilutionin

distilled

water

andboiling10min

lktA

Sites

X6mm,40%;sites

o6mm,40%;healthysites,20%

EOP,early-onsetperiodontitis;Ch.P,chronicperiodontitis;Ag.P.,aggressiveperiodontitis;ging.,gingivitis;mod.perio.,moderateperiodontitis;sev.perio.,severeperiodontitis;lkt,leukotoxin;cdt,cytolethal

distendingtoxin.


Table2.Prevalence

ofPorphyromonasgingivalisusingPCR

technique

Reference

Studygroup

Sam

plingmethod

DNA

extraction

Primers

Prevalence

Okadaet

al.(2001)


old:


10healthy,Japan

Dentalbrush

anddilution

indistilled

water

Centrifuge,

1600�

g,20min;

chem

ical


16SrRNA

Total,9.6%;healthy,4.8%;gingivitis,

9.6%;periodontitis,20%

Nozakiet

al.(2001)

31periodontitis,Japan

Paper

pointanddilution

in200ml

distilled

water

Mix

withTris/salts/Tween-20/

proteinaseK;incubation,551C;

1h;extractionwithphenol/

chloroform

/ethanol;resuspend

inTris-EDTA

Outermem

brane

protein

geneOMP

40kDaandhybridization

Pre-treatment,80%;post-treatment,

48.4%

Takeuchiet

al.(2001)

123subjects:

20healthy,

103periodontitis:

38aggressive,

65chronic,Japan

Paper

pointanddilution

1mldistilled

water

Centrifuge,

10,000r.p.m

.,5min

andresuspendpelletin

200ml

water;boiling,10min

16SrRNA

Healthy,10%;chronic

periodontitis,

95.3%;aggressiveperiodontitis,84.2%

Amanoet

al.(1999)


Curetteanddilution

in1mlPBS

Centrifuge,

12,000r.p.m

.,1min;

extractionwithkitPuregene;

resuspended

in100ml

distilled

water

Fim

Agenotype-specific

andspecies16SrRNA

Prevalence,78.5%;predominance

of

genotypes

IIandIV

Amanoet

al.(2000)

519subjects:

380healthy,


Curetteanddilution

1mlPBS

Centrifuge,

12,000r.p.m

.,1min;

extractionwithkitPuregene;

resuspended

in100ml

distilled

water

Fim

Agenotype-specific

andspecies16SrRNA

Prevalence:36.3%,healthy(genotypes

IandV);87.1%,periodontitis(genotypes

IIandIV

)Yuan

etal.(2001)

246periodontitissubjects:

105non-insulin-dependentdiabetes,

141nodiabetes,Taiwan


200ml

TE/TritonX-100

Boiling,10min;centrifuge,

10,000r.p.m

.,3min;store

supernatant

Fim

APrevalence:46.7–66.7%,site;no

differencesbetweengroups

Choiet

al.(2000)

29severeperiodontitis,

20healthy,Korea

Paper

pointand1mlRTF

Dilution,100ml

andcentrifuge,

13,000r.p.m

.,10min;

resuspended

inTE/SDS

Consensusbacterial

16S

rRNA

andhybridization

withspecificoligos

Periodontitis,100%;healthy,30%

McC

lellan

etal.

(1996)


old,USA

Paper

point

Extractionwithcommercial

kit

Interm

ediate

regionrRNAs

(ISR);nestedPCR

Prevalence,37%;noracial

orgender

differences

Tuite-McD

onnell

etal.(1997)

101families,564subjects,USA

Paper

point


kit

Interm

ediate

regionrRNAs

(ISR);nestedPCR

Prevalence,37.1%

Griffen

etal.(1999)

311subjects:

181healthy,

130periodontitis,USA

Paper

point


kit

Interm

ediate

regionrRNAs

(ISR);nestedPCR

Healthy,25%;periodontitis,79%

Griffen

etal.(1998)

311subjects:

181healthy,

130periodontitis,USA

Paper

point


kit

Interm

ediate

regionrRNAs

(ISR);nestedPCR

More

virulentstrainsin

theperiodontitis

patients,accordingto

differencesin

ISR

sequences

Umedaet

al.(1998a)

199subjects:

52Caucasian,

49Afroam

erican,48Asian,

50Hispanic,USA

Paper

point

Dilutionin

500ml

distilled

water

anddisperse

invortex;centrifuge

andresuspended

pellettwice;

store

supernatant

16SrRNA

Prevalence:Caucasian,Afroam

erican,

Asian,Hispanic

healthy/ging.:15.4%,0%,

41.2%,42.9%;initialperio.:23.1%,

61.1%,54.5%,75%;mod.perio.:50%,

4.3%,83.3%,87.5%;sev.perio.:50%,

69.2%,75%,83.3%

Darbyet

al.(2000)

57periodontitispatients:

33severeperiodontitis,

24generalized

EOP,United

Kingdom

Curetteanddilution

500ml

TE

Mix

withTE/TritonX-100boiling,5min

Fim

ASevereperiodontitis,54.5%;generalized

EOP,2.5%

Doungudomdacha

etal.(2001)

50adultperiodontitis,

United

Kingdom

Curetteanddilution

700ml

RTF

Centrifugeandresuspended

pelletin

250ml

distilled

water

Fim

AAffectedsites,93.9%

Mullally

etal.(2000)

42EOPpatients:25localized,

17generalized,Ireland

Curetteanddilution

500ml

PBS

Centrifuge,

14,000r.p.m

.,5min

andresuspended

pellet

in500ml,PBSthreetimes

16SrRNA

Prevalence,16.7%;16%

localizedEOP;

17.6%

generalized

EOP

Kam

maet

al.(2001)

16post-treatment(m

echanical

and

antimicrobialtherapy)patients

insupportivephase,

Greece

Paper

point

Dilutionin

500ml

TEanddisperse

invortex;mix

250ml

suspended

in250ml

water;centrifugeandresuspended

pellettwice;

boiling,10min;centrifuge

andstore

supernatant

16SrRNA

Prevalence:activesites,1.9%;non-active

sites,37.5%

Avila-Cam

pos&

Velasquez-M

elendez

(2002)

100subjects:

50periodontitis,

50healthy,Brazil

Paper

pointanddilution

inVMGA

IIImedium

Mix

sample

with500ml

water;

centrifugeandresuspended

pellettwice;

boiling,10min

16SrRNA

Prevalence:healthy,66%;periodontitis,

78%

Fujise

etal.(2002)

104subjects:

(1149sites)

Japan

Paper

point

Dilutionin

water

andboiling10min

Gen

PrtC

Sites

X6mm,87%;Sites

o6mm,79%;

healthysites,38%

EOP,early-onsetperiodontitis;ging.,gingivitis;mod.perio,moderateperiodontitis;sev.perio,severeperiodontitis.

1042 Sanz et al.

Table3.Prevalence

ofTannerella

forsythensisusingPCR

technique

Reference

Studygroup

Sam

plingmethod

DNA

extraction

Primers

Prevalence

Okadaet

al.(2001)


old:


10healthy,Japan

Dentalbrush

and

dilutionin

distilled

water

Centrifuge,

1600�

g,20min;

chem

ical


16SrRNA

Healthy,33.3%;gingivitis,63.9%;

periodontitis,58.3%

Choiet

al.(2000)

29sev.perio.,20healthy,

Korea

Paper

pointand1ml

RTF

Dilution,100ml

andcentrifuge,

13,000r.p.m

.,10min;

resuspended

inTE/SDS

Bacterial

common16SrRNA

Periodontitis,96.6%;healthy,55%

Tan

etal.(2001a)

160subjects:

74healthy,

86adultperiodontitis,

Singapore

Curetteanddilution,

500ml

RTF

Centrifugeandresuspended

pelletin

200ml

RTFandglass

beads;

incubation,941C,10min

Specifical16SrRNA,

prtH

gene

Prevalence:periodontitis,91%;

healthy,45%;prevalence

of

genotypeprtH:periodontitis,85%;

healthy,10%

Umedaet

al.(1998a)

199subjects:

52Caucasian,

49Afroam

erican,48Asian,

50Hispanic,USA

Paper

point

Dilutionin

500ml

distilled

water

anddisperse

invortex;centrifuge

andresuspended

pellettwotimes;

store

supernatant

16SrRNA

Prevalence:Caucasian,Afroam

erican,

Asian,Hispanic:healthy/ging.:46.2%,

50%,35.3%,50%;initialperio.:46.2%,

44.4%,45.5%,37.5%;mod.perio.:83.3%,

78.6%,66.7%,75%;sev.perio.:85.7%,

84.6%,87.5%,100%

Leyset

al.(2002)

293subjects:

121periodontitis,

USA

Paper

point

Intergenic

rRNA

region

34%

healthy,83%

periodontitis

Darbyet

al.(2000)

57periodontitis:

33severe/

periodontitis,24generalized

EOP,United

Kingdom

Curetteanddilution,

500ml

TE

Mix

withTE/Triton

X-100;boiling,5min

16SrRNA

Periodontitissevere,

21%;generalized

earlyonset,22%

Mullally

etal.(2000)

42EOP:25localized,

17generalized,Ireland

Curetteanddilution,

500ml

PBS

Centrifuge,

14,000r.p.m

.,5min;resuspended

pellet

in500ml

PBSthreetimes

16SrRNA

Prevalence,78.6%;localizedperiodontitis,

76%;generalized

periodontitis,82.4%

Kam

maet

al.(2001)

16post-treatment(m

echanical

andantimicrobialtherapy)

patients

insupportivephase,

Greece

Paper

point

Dilutionin

500ml

TEand

disperse

invortex;mix

250ml

suspended

and250ml

water;


pellettwice;

boiling,10min;

centrifugeandstore

supernatant

16SrRNA

Prevalence:activesites,87.5%;non-active

sites,68.8%

Avila-Cam

pos&

Velasquez-M

elendez

(2002)

100subjects:

50periodontitis,

50healthy,Brazil

Paper

pointanddilution

inVMGA

IIImedium

Mix

sample

with500ml

water;


pellet

twice;

boiling,10min

16SrRNA

Prevalence:healthy,30%;periodontitis,82%

Fujise

etal.(2002)

104subjects(1149sites),Japan

Paper

point

Dilutionin

distilled

water

and

boiling10min

16SrRNA

Sites

X6mm,96%;siteso6mm,86%;

healthysites,70%

Ging.,gingivitis;mod.perio.,moderateperiodontitis;sev.perio.,severeperiodontitis.


reference standard in periodontal micro-biology.

Summary

Although different methods of micro-bial diagnosis have been used for thedetection and quantification of putativeperiodontal pathogens in subgingivalsamples, there is not a single one thathas demonstrated ideal characteristics.The choice of the optimal microbial testfor adjunctive diagnostic aid in perio-dontics should be based on the follow-ing parameters:

1. Sensitivity and specificity of the test.Real-time PCR technology hasdemonstrated a high degree of sensi-tivity and specificity, mostly whencompared with standard culturing.Qualitative PCR tests, althoughhighly sensible and specific, do notprovide accurate information on thenumber of bacteria identified, and thisinformation might be crucial inclinical diagnosis. Research evidence,although limited, clearly shows anassociation between a higher bacter-ial count and disease occurrence andseverity. The mere presence of abacterial pathogen has, therefore,limited value as an adjunct to clinicaldiagnosis and treatment planning.

2. Availability of use and cost. Culture,checkerboard analysis and real-timequantitative PCR require sophisti-cated laboratory facilities and arelabour intensive, but makes themexpensive for routine clinical diag-nostic use. Standard PCR tests andother easy-to-use periodontal micro-biological methods based on immuneor molecular technologies have lim-ited capability for accurate quantifi-cation and are limited to the targetbacteria, which limits their diagnos-tic validity for clinical use.

3. Information provided. Only bacterialculturing enables the study of anti-bacterial susceptibilities and detec-tion of unexpected bacteria, whilePCR and other molecular techniqueswould allow the detection of non-cultivable microorganisms.

4. To date, there is no ideal microbialdiagnostic for adjunctive clinical usein periodontics. Bacterial culturingstill remains the gold standard.However, improved quantitativePCR technology may have an impor-tant role in the future, once it has

been fully validated with well-designed clinical trials and theircosts have been reduced with moreavailable technology.

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Address:

Mariano Sanz

Faculty of Odontology

Universidad Complutense

Plaza. Ramon y Cajal

s/n 28040 Madrid

Spain

Fax: 134-91-394-19-10

E-mail: [email protected]


Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and...

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