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Article history:Received 22 May 2009

Many methods to assess caries risk have been developed. Mostof the tests believed to predict future caries relate to the classic

have been developed to predict future caries attack [911].However, available test kits are expensive and have limitations ofidentifying a specic bacterium only. Assessing the collectiveactivity of cariogenic bacteria from a special selective nutrientmedium is therefore needed. Among available caries activity testkits is the Cariostat [12]. The test assesses acid-production ofmicroorganisms in a plaque sample inoculated in the ampoule. TheCariostat method is a caries risk assessment test used in Japan inclinical and epidemiological studies. It is based on color changes of

* Correspondence to: Omar Marianito Maningo Rodis, Department of BehavioralPediatric Dentistry, Okayama University Graduate School of Medicine and Dentistry,2-5-1 Shikata-cho, Okayama City 700-8525, Japan. Tel.: 81 (86) 235 6715; fax:81 (86) 235 6719.

Contents lists availab

Molecular and C

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Molecular and Cellular Probes 23 (2009) 259263E-mail address: omarodis@md.okayama-u.ac.jp (O.M.M. Rodis).1. Introduction

The mixed dentition period occurs when the child is in theelementary school years. It is characterized by bone growth anddevelopment, tooth exfoliation and eruption, change in dietaryhabits, early caries, frequent increase and decrease of caries activitythat occurs around the elementary school period, or when the childis about 612 years of age. During this period, caries activity isknown to have different intensity peaks, which makes cariesassessment difcult and caries prevention important [1]. Sinceearly caries and tooth loss in itself can be a sufcient predictor ofcaries attack in the future, caries risk assessment is thereforeimportant during this period [2].

factors of dental caries model: the host, cariogenic bacteria anddiet. This includes age, socio-economic factors, past caries experi-ence and presence of a specic bacterium. In general, most of thesetests are better at selecting those who will not develop caries(specicity) than they are at selecting thosewhowill develop caries(sensitivity). Recent developments in bacterial strain identicationat the molecular level have paved the way to more reliable results.

Studies have established the presence of Mutans Streptococci(MS) and Lactobacilli (LB) species as the main etiologic factors fordental caries [35]. The bacterial species most frequently isolatedfrom the mouth are Streptococcus mutans, Streptococcus sobrinus,Streptococcus salivarius [6,7] Lactobacillus casei, Lactobacillus plan-tarum and Lactobacillus fermentum [8]. Microbiological methodsAccepted 22 May 2009Available online 30 June 2009

Keywords:Caries riskCariogenic bacteriaCariostatDNAPCR0890-8508/$ see front matter 2009 Elsevier Ltd.doi:10.1016/j.mcp.2009.05.001The aim of the study was to assess the presence of six common cariogenic bacteria from Cariostat-inoculated plaque samples of Japanese elementary school children through PCR analysis and check itsassociations with caries risk testing the validity of Cariostat as a caries risk assessment tool. Thisepidemiological school-based study investigated plaque samples of 399 Japanese elementary schoolchildren. Assessed using the Cariostat, 48.2% of the children had high caries risk. DNA detection ofStreptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Lactobacillus casei, Lactobacillusplantarum, Lactobacillus fermentum and both S. mutans and S. sobrinus was seen in 65.2%, 24.1%, 69.7%,17.5%, 7.8%, 19.3%, and 17.3% of the participants, respectively. Except for S. salivarius, the presence of allother investigated bacteria resulted in a statistically signicant increase among the proportion of subjectswith high caries risk. Caries risk assessed using Cariostat was signicantly inuenced by the presence ofcariogenic bacteria. Being a selective medium for cariogenic bacteria, the Cariostat can be a useful anddirect source of cariogenic bacterial DNA for PCR analysis while effectively assessing caries risk.

2009 Elsevier Ltd. All rights reserved.a r t i c l e i n f o a b s t r a c tCulture-based PCR analysis of plaque sato assess the presence of six common cwith caries risk

Omar M.M. Rodis a,*, Seishi Matsumura a, NaoyukiSagiri Ogata b, Daniel R. Reimann c

aDepartment of Behavioral Pediatric Dentistry, Okayama University, Okayama City, JapbHospital of Medicine and Dentistry, Okayama University, Okayama City, JapancDepartment of Prosthetic Dentistry, University Medical Center Hamburg-Eppendorf, HAll rights reserved.ples of Japanese school childreniogenic bacteria and its association

riya b, Yoshihide Okazaki b,

urg, Germany

le at ScienceDirect

ellular Probes

sevier .com/locate/ymcpr

d Cethe test solution brought about by a decrease in pH caused by acidproducing microorganisms from the patients plaque. After scoring,the ampoule is shown to the patient with the intent of giving thema simple visual aid regarding their caries risk. Okazaki et al.reported that students in Japan were found to develop tooth decaythree years after being assessed as high risk by the Cariostatmethodwhen theywere 6 years old [13]. Other longitudinal studiesreported similar ndings, further validating the predictive ability ofthe Cariostat [14,15]. Though the test has been shown to havepredictive ability, less is known about its association with thepresence of cariogenic bacteria through DNA detection. If the testcorrectly assesses caries risk, cariogenic bacteria should be foundmore often in subjects assigned as having a high caries risk than inthose with low risk. It was, therefore, necessary to investigate theassociation between cariogenic bacteria and caries risk assessmentusing Cariostat.

The study aims to assess the presence of six common cariogenicbacteria from Cariostat-inoculated plaque samples of Japaneseelementary school children through PCR analysis, and to determinethe validity of the Cariostat test as a caries risk assessment tool.

2. Materials and methods

2.1. Study population

This study was part of a school health activity program, whichstarted in 1987, conducted in April 2004 with an initial list of 406elementary school children of Kobe City, Japan. Their age rangedfrom six to twelve years of age. There were seven participants whohad consented but were absent during the day of the healthexamination, which resulted in a nal count of 399 participants(219 boys and 180 girls). Plaque sampling was performed for eachgrade level: from Grade 1 (n 72; 42 boys, 36 girls), Grade 2(n 64; 42 boys, 22 girls), Grade 3 (n 73; 37 boys, 36 girls), Grade4 (n 75; 41 boys, 34 girls), Grade 5 (n 65; 34 boys, 31 girls),through Grade 6 (n 44; 23 boys, 21 girls). The students andparents were made aware of the health activity program throughthe schools newsletters while parents were requested to giveconsent with regards to their childrens participation prior to thescheduled examination. The privacy concerns of the participantswere addressed by the authors based on the schools ethicalguidelines regarding their students privacy rights. These guide-lines have been followed since the beginning of the annual healthexamination program. The program staff from the Department ofBehavioral Pediatric Dentistry of Okayama University is usuallycomposed of six to seven dentists. Dental status was not included inthe analysis due to the diverse state of tooth eruption, exfoliationand caries during the mixed dentition period.

2.2. Plaque sampling and caries risk assessment

A sterile cotton swab was used to collect samples from thebuccal and labial surfaces of the upper teeth in a continuous side toside sweeping stroke that is repeated 3 times for both quadrants.The samples were then brought back to the university in a 4 Ccontainer on the same day and incubated immediately. After 48 hof incubation at 37 C, the samples were scored by two well-trained examiners whowere calibrated prior to the scoring session.Being a colorimetric test, scores are assigned according to colorchanges after the 48-h incubation period. A score of 0 (blue; pH6.10.3) and 1.0 (green; pH 5.4 0.3) is considered as low riskwhile a score of 2.0 (yellow-green; pH 4.7 0.3) and 3.0 (yellow;pH 4.0 0.3) is considered as high risk. The inter-examineragreement rate using the 4-scale Cariostat scoring system had

O.M.M. Rodis et al. / Molecular an260a kappa value of 0.94.2.3. DNA extraction and PCR

After scoring, the samples were immediately prepared forbacterial DNA extraction and stored at -20 C until ready for PCRanalysis. In the DNA extraction procedure, the Cariostat sample wasintermittently agitated by vortexing (Ecan Tube Mixer, TM-2000,Asahi Techno Glass, Japan) for 10 s to dislodge bacteria sticking ontothe cotton swab. Bacterial pellet was produced by transferring 1 mlof the Cariostat sample to a 1.5 ml mini centrifuge tube, centrifugedat 4700 g (7500 rpm, Himac CF15R with T15AP21 rotor, Hitachi)for 10 min. DNA extraction was done using Qiagens protocol forDNA extraction of Gram-positive bacteria. The PCR (iCycler DualBlock Thermal Cycler, Biorad) method was performed with a 20 mlreaction volume of which a uniform DNA template of 2.0 ml wasused for all samples. To rule out the chance for false negative results,analysis with 16S rRNAuniversal primers conrmed the presence ofbacteria in the plaque samples [16]. Bacterial 16S rRNA is known tobe highly conserved within the bacterial species and is consideredto be a standard in bacterial identication and detection.

DNA detection was done for the following bacterial strains:S. mutans, S. sobrinus, S. salivarius, L. casei, L. plantarum, L. fermentumand both S. mutans and S. sobrinus (henceforth referred to asS. mutsob). For the mutans streptococcal strains, a 2-primer pairwas used to specically detect S. mutans and S. sobrinus at the sametime in one PCR reaction while another species-specic primerwas used to identify S. salivarius [1719]. Also, primers designed tospecically identify lactic acid bacteria including L. fermentum,L. casei and L. plantarum, were used [20,21]. Reference strains(S. mutans ATCC 25175, S. sobrinus ATCC 33478, S. salivarius ATCC7073, L. casei ATCC 4646, L. plantarum ATCC 8014, and L. fermentumATCC 14931) of the six bacteria were also included in each proce-dure as positive controls while pure distilled water (Gibco Ultra-PURE Distilled Water, Grand Island, NY, USA) was used as negativecontrol. Amplied products were subjected to electrophoresis on1% ethidium bromide-stained agarose gel in a Tris-acetate-EDTAbuffer. Products were checked at least two times under uniformconditions in accordance with the presence or absence of DNAbands and veried again randomly to conrm the results.

2.4. Data analyses

Cariostat scores and presence of bacteria were graphically pre-sented as proportions for all participants stratied by grade level.Differences in Cariostat scores, caries risk, and presence of bacteriawith respect to gender and grade level were tested for proportions(caries risk, presence of oral bacteria) using chi2-test and for rankeddata (Cariostat scores) using Wilcoxon rank-sum test (two-groupcomparison) and KruskallWallis rank test (more than two-groupcomparison).

To analyze the association between presence of cariogenicbacteria with Cariostat scores and with caries risk [low (Cariostatscores 0 and 1.0) and high (Cariostat scores 2.0 and 3.0)], severaldata analyses were performed. First, the functional relationships,i.e., the linear or non-linear relationship between a predictor(presence of cariogenic bacteria) and the outcome (Cariostat scoreand caries risk) was explored. For approximate linear relationshipsbetween presence of cariogenic bacteria and the categorizedmeasures (scores 0, 1.0, 2.0, and 3.0) of the Cariostat test, wecomputed polychoric correlation coefcients. To assess the corre-lation between presence of cariogenic bacteria and caries risk,a tetrachoric correlation coefcient was computed.

To compare the prevalence of cariogenic bacteriawith caries riskassessed using Cariostat, prevalence rate ratios (PRRs) werecomputed. These measures are similar to risk ratios, for example,

llular Probes 23 (2009) 259263a PRR of 1.4 for S. mutanswould indicate that the prevalence of high

All analyses were performed using the statistical softwarepackage STATA (Stata Statistical Software: Release 9.2 StataCorp,College Station, TX), with the probability of a type I error set at the0.05 level.

3. Results and discussion

3.1. Distribution of caries risk and prevalence of cariogenic bacteria

The distribution of Cariostat scores and DNA detection accordingto grade levels is presented in Figs. 1 and 2. High caries risk wasseen in 48.2% of the participants (Cariostat score 2.0 and 3.0). InitialPCR analysis with universal primers conrmed the presence ofbacteria in all 399 plaque samples. DNA band detection of S. mutans,S. sobrinus, S. salivarius, L. casei, L. plantarum, L. fermentum andS. mutsob was seen in 65.2%, 24.1%, 69.7%, 17.5%, 7.8%, 19.3% and

Fig. 1. Proportion of children with a particular Cariostat score.

O.M.M. Rodis et al. / Molecular and Cellular Probes 23 (2009) 259263 261caries risk is 1.4 times more likely (equals a 40% increase) inpatients with the presence of S. mutans than in patients without S.mutans. PRRs were computed using general linear models (GLM)with binominal distribution and iterated, reweighted least-squares(IRLS) optimization of the deviance without and with adjustmentfor grade level and gender.

In the nal step of the analysis, the clinical usefulness of theCariostat test used as a diagnostic test to determine the presence ofcariogenic bacteria was assessed. The classication by PCR ofpatients status regarding the presence of cariogenic bacteria wasconsidered the reference standard, while diagnostic test accuracymeasures such as sensitivity, specicity, and positive and negativepredictive values including 95% condence intervals were calcu-lated. In addition, the positive and negative likelihood ratiosincluding their 95% condence intervals were calculated. A positivelikelihood ratio describes the ratio of the probability of the positivetest result among patients with the disease to the probability inpatients who do not have the disease, whereas the negative like-lihood ratio describes the ratio of the probability of the negativetest result among patients with the disease to the probability inpatients who do not have the disease [22]. Likelihood ratio can beused to estimate post-test odds by multiplying the pre-test odds bythe likelihood ratio [23]. Guidelines for the goodness of diagnostictests exist andwere applied to judge Cariostat as a diagnostic test todetect the presence of cariogenic bacteria [24]. According to theserecommendations, likelihood ratio values of more than 10 or lessthan 0.1 signify convincing diagnostic evidence, values of 510 and0.10.2 signify high diagnostic evidence, values of 25 and 0.20.5signify weak diagnostic evidence and values between 2 and 0.5signify hardly relevant diagnostic evidence.Fig. 2. Proportion of detected cario17.3% of the participants, respectively. There was no signicantdifference in Cariostat scores (Wilcoxon rank sum: P> 0.05) andin caries risk (chi2-test: P> 0.05) between genders. S. mutans wassignicantly more commonly observed among girls than boys(72.2% vs. 59.4%, chi2-test: P< 0.01) whereas S. salivarius wasmore prevalent among boys than girls (62.8% vs. 75.3%; chi2-test:P< 0.01). Neither S. sobrinus, L. casei, L. plantarum, L. fermentum norS. mutsob differed signicantly with respect to gender (chi2-test: allP> 0.05). There was neither a signicant effect of grade level onCariostat scores (KruskallWallis rank test: P> 0.05) nor on cariesrisk (chi2-test: P> 0.05). The presence of S. sobrinus (chi2-test:P< 0.001), S. salivarius (chi2-test: P< 0.001), L. plantarum (chi2-test: P< 0.05) and S. mutsob (chi2-test: P< 0.001) differed signi-cantly among grade levels, but not the presence of S. mutans,L. casei, and L. fermentum (chi2-test: all P> 0.05).

DNA detection of common oral bacteria known to cause dentalcaries has been associated with a high score of Cariostat-assessedcaries risk in this sample of school children. The presence of thesebacteria early in childhood leads to caries, which may cause earlyloss of teeth. To prevent caries in the permanent dentition, caremust be taken for the deciduous teeth to be caries-free until thetime of exfoliation. In the study, we found signicant differences inthe distribution of cariogenic bacteria among gender and gradelevels for some bacteria. However, there was no signicant differ-ence in Cariostat scores or caries risk among grade levels or gender.Among the most commonly detected cariogenic bacteria in the oralcavity included in this study, S. salivarius, S. mutans and S. sobrinuswere found to be the most prevalent bacteria detected throughDNA in this group of children. Hamada and Slade reported thatS. salivarius has been known to inhabit the mouth 24 h after birthgenic bacteria per grade level.

Table 1Proportions of subjects with high caries risk; prevalence rate ratios (PRRs) including their 95% condence interval (95% CI) and level of statistical signicance for the inde-pendent variables (presence of cariogenic bacteria) on the dependent variable (high caries risk) and adjusted for grade level and gender.

High caries risk [%] PRR (95% CI) P-value PRR (95% CI) gradeand gender adjusted

P-value grade andgender adjusted

S. mutans No 35.3 1.56 (1.212.00) 0.05), L. plantarum rpolychoric 0.34 (P> 0.05), L. fermentumrpolychoric 0.70 (P< 0.05) and S. mutsob rpolychoric 0.25 (P> 0.05).Correlation coefcients for the relationship of caries risk with thepresence of cariogenic bacteria were: for S. mutans rtetrachoric 0.30(P< 0.001), S. sobrinus rtetrachoric 0.20 (P< 0.05), S. salivariusrtetrachoric0.14 (P> 0.05), L. casei rtetrachoric 0.27 (P< 0.01),

Table 2Presence (Pres) of cariogenic bacteria; sensitivity (Sens), specicity (Spec), and positive (presence of cariogenic bacteria; positive (LR) and negative likelihood ratio (LR); for a

Diagnostic test accuracy measures for Cariostat to diagnose t

Pres (95% CI) Sens (95% CI) Spec (95% CI)

S. mutans 65.2 (60.369.8) 55.0 (48.761.2) 64.8 (56.272.7)S. sobrinus 24.1 (20.028.6) 58.3 (47.868.3) 55.1 (49.360.8)S. salivarius 69.7 (64.974.2) 45.2 (39.451.4) 45.5 (36.454.8)L. casei 17.5 (13.921.6) 64.3 (51.975.4) 55.3 (49.860.8)L. plantarum 7.8 (5.310.9) 80.7 (62.592.6) 54.6 (49.459.8)L. fermentum 19.3 (15.423.5) 93.5 (85.597.9) 62.7 (57.268.0)S. mutans & S. sobrinus 17.0 (14.021.4) 62.3 (49.873.7) 54.8 (49.360.3)L. plantarum rtetrachoric 0.45 (P< 0.001), L. fermentum rtetrachoric 0.78 (P< 0.001) and S. mutsob rtetrachoric 0.24 (P< 0.05). Thesendings were supported by regression analyses (GLM) comparingthe prevalence of high caries risk in subjects with and without thepresence of various cariogenic bacteria (Table 1). Using the preva-lence of the cariogenic bacteria as independent variables, thepresence of all other investigated cariogenic bacteria, except for S.salivarius, resulted in a statistically signicant increase in theproportion of subjects with high caries risk assessed using theCariostat test (Table 1). Additionally, regression analysis wasadjusted for grade level and gender. However, results didnt changesubstantially (Table 1).

A highly signicant relationship was observed between thepresence of S. mutans, L. plantarum, L. casei and L. fermentum andcaries risk assessed using the Cariostat method. All four are highlyacidogenic and aciduric, which are oral bacterial properties thathasten tooth demineralization. Another very interesting nding ofthis study was the high association of L. fermentumwith Cariostat-assessed caries risk. However, there are still no studies of the directassociation of L. fermentum with dental caries in children. Thus,further studies of L. fermentum must be done to assess its associa-

1.78)

were high (80.7% and 93.5%) whereas for bacteria that were more References

O.M.M. Rodis et al. / Molecular and Cellular Probes 23 (2009) 259263 263prevalent (S. mutans and S. salivarius), values for sensitivity werelow (55.0% and 45.2%). Values for specicity were low for allbacteria and ranged from 45.5% to 64.8%, i.e., only up to two thirdsof the subjects not having the specic bacterium were correctlyidentied using the Cariostat test. The positive predictive valuesranged from 13.0% to 74.5% and the negative predictive valuesranged from 26.6% to 97.6%. High caries risk assessed using Cario-stat increased the probability of actually having cariogenic bacteriafor almost all investigated bacteria (Table 2), e.g., for S. mutans,a high caries risk increased the probability of having this bacteriumfrom 65.2% (prevalence of S. mutans, i.e., pre-test probability) to74.5% (post-test probability or positive predictive value) or forpresence of S. mutsob, from 17.0% to 22.4%. This is equivalent toa positive likelihood ratio (LR) of 1.56 and 1.38, respectively(Table 2). The values for LR varied from 0.83 to 2.51 across thedifferent bacteria. Almost all of these values are considered hardlyrelevant for a diagnostic test. Only for L. fermentum can the test beconsidered as having weak diagnostic evidence. Except forS. salivarius, the negative likelihood ratio (LR) for all bacteria wasless than 1 and varied from 0.10 to 0.76, i.e., the presence of bacteriaamong subjects with low caries risk was lower in comparison withthe presence of bacteria among high caries risk subjects. Only theLR of L. plantarum reached the value of 0.50, which is consideredto be the border for weak diagnostic evidence and the LR ofL. fermentum reached the value of 0.10, which is considered to bethe border for convincing diagnostic evidence.

Caries risk assessed using Cariostat was signicantly inuencedby the presence of cariogenic bacteria commonly found inside theoral cavity. It provides a collective representation of the entiregroup of bacteria present in a sample and not the presence of justone bacterium. The likelihood ratio analyses evaluated Cariostat asa diagnostic test for the presence of a specic bacterium in theCariostat sample. Results showed almost all values were consideredhardly relevant for a diagnostic test. These results were expectedsince the Cariostat was developed to assess the overall quality ofbacteria present in an individual. As with the factors in the cariesmodel, caries risk depends not only on one factor but a combinationof factors. Moreover, the Cariostat test was developed as a selectivemedium not only for Mutans Streptococci but for the Lactobacillispecies as well. Although dental status is widely used as an indi-cator of caries risk in the permanent dentition, it is difcult for it tobe used as such during the mixed dentition period. In a review oftwenty-eight studies evaluating the effectiveness of routine dentalchecks, there was no consistency across multiple studies in thedirection of effect of different dental check frequencies onmeasuresof caries in deciduous mixed or permanent dentition [30].

In conclusion, the study conveys the importance of cariesactivity tests that can assess caries risk by taking into considerationthe overall quality of cariogenic bacteria present and not just thepresence of a specic bacterium. Although some studies haveshown the validity of Cariostat through clinical and epidemiologicalstudies, this study is the rst one that has checked the presence ofsix commonly found oral bacteria through DNA detection fromCariostat-inoculated plaque samples.

Acknowledgements

The authors wish to acknowledge the assistance of the principal,teachers and students of the elementary school in Kobe, Prof.Tsutomu Shimono, initiator of the school program, and Dr. DuanChunyan, Department of Stomatology, Dalian University, China.[1] Schlagenhauf U, Rosendahl R. Clinical and microbiological caries-risk param-eters at different stages of dental development. J Pedod 1990;14(3):1413.

[2] NIH Consensus Statement. Diagnosis and Management of Dental CariesThroughout Life 2001;18(1):124.

[3] Narhi TO, Kurki N, Ainamo A. Saliva, salivary micro-organisms, and oral healthin the home-dwelling old elderly a ve-year longitudinal study. J Dent Res1999;78(10):16406.

[4] Loesche WJ, Schork A, Terpenning MS, Chen YM, Stoll J. Factors which inu-ence levels of selected organisms in saliva of older individuals. J Clin Microbiol1995;33(10):25507.

[5] Brambilla E, Twetman S, Felloni A. Salivary mutans streptococci and lactoba-cilli in 9- and 13-year-old Italian schoolchildren and the relation to oral health.Clin Oral Investig 1999;3(1):710.

[6] Whiley RA, Beighton D. Current classication of oral Streptococci. OralMicrobiol Immunol 1998;13:195216.

[7] Thurnheer T, Gmur R, Giertsen E, Guggenheim B. Automated uorescent insitu hybridization for the specic detection and quantication of oral strep-tococci in dental plaque. J Microbiol Meth 2001;44:3947.

[8] Marchant S, Brailsford SR, Twomey AC, Roberts GJ, Beighton D. The predom-inant microora of nursing caries lesions. Caries Res 2001;35:397406.

[9] Kohler B, Pettterson BM, Bratthall D. Streptococcus mutans in plaque andsaliva and the development of caries. Scand J Dent Res 1981;89:1925.

[10] Jordan HV, Laraway R, Snirch R, Marmel M. A simplied diagnostic system forcultural detection and enumeration of Streptococcus mutans. J Dent Res1987;66:5761.

[11] Klock B, Krasse B. A comparison between different methods for prediction ofcaries activity. Scand J Dent Res 1979;87:12939.

[12] Shimono T, Sobue S. A new colorimetric caries activity test. Dent Outlook1974;43(6):82935.

[13] Okazaki Y, Ji Y, Oyuntsetseg B, Rodis O, Hori M, Matsumura S, Shimono T. Levelof caries activity and an estimate in the increase of permanent teeth caries:a three-year follow up study in preschool senior children. Ped Dent J2005;15(1):15.

[14] Nishimura M, Bhuiyan MM, Matsumura S, Shimono T. Assessment of the cariesactivity test (Cariostat) based on the infection levels of mutans streptococciand lactobacilli in 2 to 13-year-old childrens dental plaque. ASDC J Dent Child1998;65(4):24851.

[15] Tsubouchi J, Yamamoto S, Shimono T, Domoto PK. A longitudinal assessment ofpredictive value of a caries activity test in young children. J Dent Child1995;34:347.

[16] Sato T, Hu JP, Ohki K, Yamaura M, Washio J, Matsuyama J, Takahashi N. Rapididentication of cariogenic bacteria by 16S rRNA genes PCR-RFLP analysis.Cariol Today 2001;2:813.

[17] Igarashi T, Yamamoto A, Goto N. Direct detection of the Streptococcus mutansin human dental plaque by polymerase chain reaction. Oral MicrobiolImmunol 1996;11:2948.

[18] Igarashi T, Yamamoto A, Goto N. PCR for detection and identication ofStreptococcus sobrinus. J Med Microbiol 2000;49:106974.

[19] Igarashi T, Yano Y, Yamamoto A, Sasa R, Goto N. Identication of Streptococcussalivarius by PCR and DNA probe. Lett Appl Microbiol 2001;32:3947.

[20] Chagnaud P, Machinis K, Coutte LA, Marecat A, Mercenier A. Rapid PCR-basedprocedure to identify lactic acid bacteria: application to six commonLactobacillus species. J Microbiol Meth 2001;44:13948.

[21] Dickson EM, Riggio MP, Macpherson L. A novel species-specic PCR assay foridentifying Lactobacillus fermentum. J Med Microbiol 2005;54:299303.

[22] McGee S. Simplifying likelihood ratios. J Gen Intern Med 2002;17:64750.[23] Deeks JJ, Altman DG. Diagnostic tests 4: likelihood ratios. BMJ 2004;329:

1689.[24] Jaeschke R, Guyatt GH, Sackett DL. Users guide to medical literature. III. How

to use an article about a diagnostic test. B. What are the results and will theyhelp me in caring for my patients? The Evidence-Based Medicine WorkingGroup. JAMA 1994;271(9):7037.

[25] Hamada S, Slade HD. Biology, immunology and cariogenicity of Streptococcusmutans. Microbiol Rev 1980;44:33184.

[26] Emilson CG, Ravald N, Birkhed D. Effects of a 12-month prophylacticprogramme on selected oral bacterial populations on root surfaces with activeand inactive carious lesions. Caries Res 1993;27(3):195200.

[27] Hirose H, Hirose K, Isogai E, Miura H, Ueda I. Close association betweenStreptococcus sobrinus in the saliva of young children and smooth-surfacecaries increment. Caries Res 1996;27:2927.

[28] Nie M, Fan M, Bian K. Transmission of mutans streptococci in adults withina Chinese population. Caries Res 2002;36:1616.

[29] Okada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, Kozai K. Longitudinalstudy of dental caries incidence associated with Streptococcus mutans andStreptococcus sobrinus in pre-school children. J Med Microbiol 2005;54:6615.

[30] Davenport CF, Elley KM, Fry-Smith A, Taylor-Weetman CL, Taylor RS. Theeffectiveness of routine dental checks: a systematic review of the evidencebase. Brit Dent J 2003;195:8798.

Culture-based PCR analysis of plaque samples of Japanese school children to assess the presence of six common cariogenic bacteria and its association with caries riskIntroductionMaterials and methodsStudy populationPlaque sampling and caries risk assessmentDNA extraction and PCRData analyses

Results and discussionDistribution of caries risk and prevalence of cariogenic bacteriaRelationship between caries risk and presence of cariogenic bacteriaUse of Cariostat as a diagnostic test for the presence of cariogenic bacteria

AcknowledgementsReferences