NIH Public Access Author Manuscript Quintessence Int. Author manuscript; available in PMC 2011 August 6.

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1 NIH Public Access Author Manuscript Published in final edited form as: Quintessence Int April ; 38(4): e213 e217. Microbial acid production (Clinpro Cario L-Pop) and dental caries in infants and children W. A. Bretz, DDS, Dr PH 1, P. M. A. Corby, DDS 2, S. Costa, DDS 3, M. Quadros, DDS 3, V. S. Tavares 4, G. Moreira 4, M. R. Melo Filho, DDS, MS 5, and R. J. Weyant, DMD, Dr PH 6 1 Associate Professor, New York University, College of Dentistry, New York, New York 2 Assistant Professor, University of Pittsburgh, School of Dental Medicine, Pittsburgh, Pennsylvania 3 Assistant Professor, Universidade Estadual de Montes Claros (UNIMONTES), Montes Claros, Brazil 4 Bachelor of Sciences, Universidade Estadual de Montes Claros (UNIMONTES), Montes Claros, Brazil 5 Assistant Professor, Universidade Estadual de Montes Claros (UNIMONTES), Montes Claros, Brazil 6 Associate Professor, University of Pittsburgh, School of Dental Medicine, Pittsburgh, Pennsylvania Abstract Objective The purpose of this study was to evaluate the diagnostic utility of the Clinpro Cario- L-Pop test as it relates to dental caries rates and severity in infants and children. Method and Materials The study population was comprised of 771 infants and children who were on average 5.2 years old (range of 1.5 to 8 years old). Examiners conducted dental caries clinical examination using established criteria. In addition, lesion severity was determined be measuring its depth. An indicator swab was applied to the tongue dorsum until completely moistened with saliva. The indicator swab was processed according to the manufacturer s instructions, and acid production was assessed with the aid of a color chart. Results Twenty-three percent of children were caries free, and 7% (n = 50) of participants were categorized as having low production of lactic acid (scores 1 to 3), 17% (n = 135) moderate production of lactic acid (scores 4 to 6), and 76% (n = 586) high production of lactic acid (scores 7 to 9). There was a tendency for moderate and high lactic acid formers to exhibit higher surfacebased caries prevalence rates, higher rates for deep dentinal lesions, and increased lesion severity. There was a linear increase of white spot surface-based lesions from low to high lactic acid formers and for initial dentinal lesions. Clinpro Cario-L-Pop test results, when controlling for age and gender, significantly distinguished caries-free participants from those exhibiting any form of decay. Conclusion These results suggest that Clinpro Cario-L-Pop test was useful in explaining elevated frequency and severity of dental caries in spite of the high levels of decay and of microbial acid production observed in this population. Correspondence: Dr Walter A. Bretz, New York University, College of Dentistry, Mail Code 9431, 345 East 24th Street, New York, NY Fax wb36@nyu.edu.

2 Bretz et al. Page 2 Keywords children; dental caries; infants; lesion severity; microbial acid production Acid production by acidogenic oral microorganisms as a result of carbohydrate bioavailability is a determinant factor in the development of dental decay. The mutans streptococci (Streptococcus mutans) have been associated mainly with human dental decay in populations throughout the world. 1 The mutans streptococci are highly cariogenic organisms in every animal model system in which they have been tested. 2 The mutans streptococci are more efficient and more diverse in their metabolism of carbohydrates, particularly sucrose, than other tested oral microorganisms; that is, they use sucrose at a faster rate per unit cell protein than other dental plaque organisms, and they exhibit optimal metabolic activity (acid production) at ph 5.0, the same ph at about which the tooth enamel dissolves. 1 Studies employing cultural methods have shown that a finite number of non mutans streptococci organisms are also acidogenic and can potentially influence dental caries formation. 3,4 More recently, with the advent of molecular analysis for bacterial identification and enumeration, it becomes possible to perform a detailed description of the microbiota associated with dental decay. In fact, studies5 employing bacterial molecular analysis have shown that a number of organisms, including Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, Streptococcus salivarius, Streptococcus constellatus, Streptococcus parasanguinis, and Lactobacillus fermentum, were significantly associated with early childhood caries. Together, these organisms can be conceivably involved in acid production leading to the initiation and progression of dental decay. A new biochemical test has become available that measures the production of lactic acid by metabolically active cariogenic bacteria (Clinpro Cario-L-Pop; 3M Espe). Thus, the higher the metabolic activity of these bacteria, the higher the potential of these bacteria to promote caries development. Early attempts to link caries diagnostic tests that measured microbial acid production have failed to adequately link the presence of pure cultures of S. mutans and lactobacilli, which are known to be acid producers, to promote color changes from blue to yellow even when high numbers of these organisms were present. 6 These tests employ a semisynthetic liquid containing sucrose, tryptose, and bromcresol yellow and purple indicators 7 that may or may not detect overall lactic acid production by a number of members of cariogenic bacteria. These tests have provided conflicting results when test results were correlated to dental caries outcomes. 6,7 Clinpro Cario-L-Pop performance is based on enzymatic oxidation of lactic acid by lactate-dehydrogenase coupled to a cascade of redox indicators that generates a blue color signal. This lactate-dehydrogenase based enzymatic reaction may therefore capture more accurately a wide range of microbial acid producers that might be involved in caries promotion, including the mutans streptococci and non mutans streptococci organisms. 1,3,4 The purpose of this study was to evaluate the diagnostic utility of the Clinpro Cario-L-Pop test as it relates to dental caries rates and severity in a large cohort of infants and children.

3 Bretz et al. Page 3 METHOD AND MATERIALS Demographic characteristics of the study population Infants and children were ascertained from a government-based health services registry. Consent was obtained form all participants following the University of Pittsburgh and Universidade Estadual de Montes Claros Institutional Review Boards guidelines. Infants and children were of a low socioeconomic level who resided in the urban setting of the city of Montes Claros, State of Minas Gerais, Brazil. This community lacks fluoride in its water supplies, and parents reported 90% of the participants having never visited a dentist. The sample consisted of 771 infants and children who were on average 5.2 (SD = 2.2) years old (range of 1.5 to 8 years old). Forty-eight percent of participants were males. Dental caries examination Dental caries variables Clinpro Cario-L-Pop test Prior to the caries exam, all participants had their teeth thoroughly brushed and flossed by dental hygienists. This was necessary for accurate clinical examinations and a requirement to execute the Clinpro Cario-L-Pop test. Two examiners conducted dental caries clinical examination according to NIDCR8 criteria modified to distinguish caries lesions with intact enamel surface (white spot lesions) from cavitated caries lesions. In addition, once a lesion was detected, its severity was determined be measuring its depth as follows: 1 white spot lesions; 2 lesions confined to enamel with loss of intact surface; 3 lesions where the dentin could be visualized at initial stages of breakdown; and 4 lesions >2 to 3 mm into dentin. Summary variables were generated for the following variables for each participant: 1. Dental caries dichotomous prevalence (DDich): This variable measured the absence or presence of at least 1 decayed surface independent of severity. 2. Surface-based caries prevalence rate (SBCPR): The total number of decayed surfaces (rank of severity that could possibly range from 1 to 4) was divided by the total number of surfaces present? SBCPRs according to lesion severity (SBCPR WS/IE/ID/DD): The total number of white spot, initial enamel, initial dentin, and deep dentin decayed surfaces separately was divided by the total number of surfaces present? SBCPRs based on lesion location (SBCPROccl/Mes/Dist/Bucc/Ling): The total number of decayed surfaces in occlusal, mesial, distal, buccal, and lingual surfaces separately was divided by the total number of surfaces present? Lesion severity index (LSI): The total number of white spot lesions? 1 + total number of initial enamel lesions? 2 + total number of initial dentinal lesions? 3 + total number of deep dentinal lesions? 4 was divided by the total number of surfaces present to derive a balanced outcome of caries severity for each participant. Infants and children had an indicator swab applied to the tongue dorsum by rotating it back and forth at least 4 times in order to generate a tongue biofilm sample. The indicator swab was processed according to the manufacturer s instructions by inserting it into a blister prototype containing the diagnostic reagents. The swab indicator was compared to a color

4 Bretz et al. Page 4 Statistical analysis RESULTS DISCUSSION chart where the levels of acid production were assessed. Test scores ranged form 1 to 9. Scores 1 to 3 indicate a low formation rate of lactic acid, 3 to 6 a medium formation rate of lactic acid, and 7 to 9 a high production of lactic acid (Fig 1). Data were submitted to regression analysis, analysis of variance, and descriptive analysis by employing SAS statistical software (SAS). The kappa statistic was used to assess interexaminer reliability for dental caries. Cronbach s alpha coefficient of reliability (or consistency) was employed to measure the average intercorrelation between 2 scorers for the Clinpro Cario-L-Pop test. These exercises were performed in 10% of the participants. All computations employed 2-tailed tests. Data for the Clinpro Cario-L-Pop test was analyzed as a continuous variable (range of scores of 1 to 9) or as a categorical variable (1 = scores of 1 to 3, 2 = scores of 4 to 6, and 3 = scores of 7 to 9). The intraexaminer kappa statistic for decayed lesions diagnosed in the enamel and dentin including white spot lesions at reliability examinations was 0.75, and the weighted kappa was Cronbach s alpha statistic revealed an excellent interscorer agreement for the Clinpro Cario-L-Pop test (C-alpha = 0.95). Twenty-three percent of participants were caries free (n = 176). Seven percent (n = 50) of participants were categorized as having low production of lactic acid (scores 1 to 3), 17% (n = 135) moderate production of lactic acid (scores 4 to 6), and 76% (n = 586) high production of lactic acid (scores 7 to 9). Table 1 shows dental caries variables by Clinpro Cario-L-Pop test categorical results. There was a tendency for moderate and high lactic acid formers to exhibit higher surface-based caries prevalence rates, higher rates for deep dentinal lesions, and increased lesion severity. There was a linear increase of white spot surface-based lesions from low to high lactic acid formers. This was also true for initial dentinal lesions. These trends, however, did not achieve statistical significance. No apparent trends were found for initial enamel lesions and lactic acid production (Table 1) and for surface-based rates by lesion location on the tooth (data not shown). We next evaluated if Clinpro Cario-L-Pop test continuous results (full range of scores from 1 to 9) controlling for age and gender could predict dental caries summary variables (data not shown). Clinpro Cario-L-Pop test results significantly distinguished caries-free participants from those exhibiting any form of decay (P <.05). Test results could not predict surface-based caries prevalence rates by lesion severity or by the overall LSI. When surfacebased caries prevalence rates were analyzed on the basis of their location, a statistically significant inverse relationship (P at least <.018) could be observed between Clinpro Cario- L-Pop test results and SBCPRs for the occlusal, distal, and buccal surfaces as judged by the negative parameter estimates. This means that the lower the SBCPRs for the occlusal surfaces, the higher the Clinpro Cario-L-Pop test scores. The Clinpro Cario-L-Pop test proved to be a reliable method for assessment of the bacterial metabolic activity (acid production), as interscorer reliability estimates were high (C-alpha = 0.95). This level of reliability was attained in 10% of a large cohort of infants and children (N = 771), which is an important feature for the validity of any diagnostic test that relies on subjective interpretation.

5 Bretz et al. Page 5 CONCLUSIONS Acknowledgments References The distribution of the results for the Clinpro Cario-L-Pop test clearly demonstrated that this cohort of infants and children had for the most part high levels of microbial acid production. This could be partially explained by absence of fluoride in water supplies in the region the cohort inhabits, underutilization of dental services, and possibly high ingestion of sucrosebased nutrients. Accordingly, this population exhibited very high levels of surface-based caries prevalence rates, that is, an average of 10% of decayed surfaces. Although a rather skewed distribution of Clinpro Cario-L-Pop test results was observed, some trends were apparent for dental caries rates (Table 1). Overall, SBCPRs, SBCPRs for white spot and dentinal lesions, and LSI rates tended to be higher in moderate and high acid formers than in low acid formers. It remains to be seen if in populations with a more normal distribution of Clinpro Cario-L-Pop test results and of caries rates and severity would strengthen associations between microbial acid production and dental caries rates. Next we evaluated the use of the Clinpro Cario-L-Pop test as an explanatory variable for all summary variables for dental caries controlled for age and gender (data not shown). Clinpro Cario-L-Pop test results were able to distinguish between caries-free and caries-active (at least 1 decayed surface) infants and children. That meant that caries-free participants had lower Clinpro Cario-L-Pop scores when compared to caries-active participants. Conversely, we found an inverse relationship between SBCPRs for specific tooth locations, such as the occlusal, distal, and buccal surfaces, and Clinpro Cario-L-Pop test results. We offer no explanation for these findings other than it is conceivable to assume that measurement of plaque acid production at discrete surface sites would more accurately correlate with caries outcomes at those specific locations. As such, a similar test governed by the same principles of the Clinpro Cario-L-Pop test would be more informative if it measured microbial sitespecific acid production and therefore could more accurately correlate with dental caries profiles at specific tooth locations. In summary, the Clinpro Cario-L-Pop test results showed association with dental caries outcomes, principally by differentiating caries-free from caries-active patients. Notably, the test was useful in explaining elevated frequency and severity of dental caries in spite of the high levels of decay and of microbial acid production observed in this population. It would be important to determine the microbial acid producing organisms that are responsible for the Clinpro Cario-L-Pop test results. More important, it would be desirable to determine the caries predictive ability of the Clinpro Cario-L-Pop test in longitudinal dental caries studies. We are currently undertaking efforts to elucidate these critical issues. 1. The Clinpro Cario-L-Pop test results were able to differentiate caries-free from caries-active patients. 2. Overall surface-based caries prevalence and lesion severity index rates tended to be higher in moderate and high acid formers than in low acid formers. 3. The Clinpro Cario-L-Pop test proved to be a reliable method to determine microbial acid production based on a high interscorer agreement. This study was partially supported by NIH/NIDCR grants DE15351 and DE Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986; 50: [PubMed: ]

6 Bretz et al. Page 6 2. Keyes PH. Research in dental caries. J Am Dent Assoc. 1968; 76: [PubMed: ] 3. Sansone C, Van Houte J, Joshipura K, Kent R, Margolis HC. The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low ph with dental caries on enamel and root surfaces. J Dent Res. 1993; 72: [PubMed: ] 4. van Houte J, Lopman J, Kent R. The final ph of bacteria comprising the predominant flora on sound and carious human root and enamel surfaces. J Dent Res. 1996; 75: [PubMed: ] 5. Becker MR, Paster BJ, Leys EJ, et al. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol. 2002; 40: [PubMed: ] 6. Camling E, Emilson C-G. Results with the caries activity test (Cariostat) compared to prevalence of mutans streptococci and lactobacilli. Swed Dent J. 1989; 13: [PubMed: ] 7. Koroluk L, Hoover JN, Komiyama K. The sensitivity and specificity of a colorimetric microbiological caries activity test (Cariostat) in preschool children. Pediatr Dent. 1994; 16: [PubMed: ] 8. Kastle L, Selwitz R, Oldakeki R. Coronal caries in the primary dentition and permanent dentition of children and adolescents 1 17 years of age: United States, J Dent Res. 1996; 75: [PubMed: ] 9. Bretz WA, Djahajah C, Almeida RS, Hujoel PP, Loesche WJ. Relationship of microbial and salivary parameters with dental caries in Brazilian pre-school children. Community Dent Oral Epidemiol. 1992; 20: [PubMed: ]

7 Bretz et al. Page 7 Fig 1. Interpretation of Clinpro Cario-L-Pop test results.

8 Bretz et al. Page 8 Table 1 Analysis of variance of dental caries summary variables by Clinpro Cario-L-Pop test results (Clinpro Cario-L- Pop results) Dental caries variable 1 (n = 50) 2 (n = 133) 3 (n = 586) SBCPR 8.1% (5.3, 10.8) * 10.4% (8.7, 12.1) 9.7% (8.9, 10.9) SBCPR WS 1.9% (0.9, 2.8) 2.2% (1.7, 2.8) 2.4% (2.1, 2.7) SBCPR IE 3.6% (2.5, 4.7) 3.8% (3.1, 4.5) 3.8% (3.4, 4.1) SBCPR ID 1.2% (0.5, 1.8) 1.7% (1.2, 2.1) 1.6% (1.4, 1.8) SBCPR DD 1.4% (0.3, 2.6) 2.7% (1.9, 3.4) 2.0% (1.6, 2.3) LSI 0.18 (0.1, 0.3) # 0.25 (0.2, 0.3) 0.22 (0.2, 0.2) * Mean % (95% confidence interval, lower, upper). # Mean (95% confidence interval, lower, upper). Microbial Acid Production (Clinpro Cario L-Pop ) and Dental Caries in Infants and Children