Comparison of Clinpro Cario L-Pop test with other conventional bacterial tests used for caries risk estimation

Transcription

1 1 Comparison of Clinpro Cario L-Pop test with other conventional bacterial tests used for caries risk estimation Ghada Nimeri Sofia Sölver Supervisors Margaret Sällberg Chen and Anette Oliveby

2 2 Authors contributions Ghada Nimeri: Writing of the summary, introduction, calibration of the CCLP test and the discussion, organizing and performing the bacterial tests on the participants. Sofia Sölver: Providing reference articles from Pub-Med and writing the material and method chapter and some of the discussions. Created the tables and graphs. Editing and layout work. Organized the practical work and performing the bacterial tests on the participants.

3 3 Summary The detection of caries risk is not easy due to the involvement of many factors in the oral cavity. The development of new quick methods for caries risk detection is desired. Clinpro Cario L-Pop (CCLP) is a new semiquantitative method that is manufactured by 3M ESPE, (Seefeld, Germany) and claimed by the manufacturer to be able to detect caries risk patients in only a few minutes. The test detects the production of lactic acid from cariogenic bacteria. Aim The aims of this study were to compare the CCLP test with known conventional standard methods for detecting caries risk patients, to evaluate the reliability of the CCLP test and to evaluate if there are any differences when the CCLP color chart is read in the light of different sources. Methods A total of 77 participants, both students and visitors were recruited from the adult dental care clinic (VUX) at the Institute of Odontology, Karolinska Institutet. The participants were divided into two groups, one caries free and one caries active group, according to criteria used at VUX. Clinical examination and anamnesis were carried out and data from salivary flow, eating habits, fluoride intake, caries activity and experience were gathered for the evaluation of caries risk. Dentocult LB, laboratory culturing of Lactobicilli (LB), and Streptococci Mutans (SM) and CCLP tests were also performed and the results were compared. Calibrations of the CCLP method was done by first reading the CCLP color chart and then evaluate the influence of different light sources. Results The results showed that there were no correlation between the known conventional methods used for caries risk detection and the CCLP test. That can be due to methodological difficulties, for instance when reading the CCLP color chart.

4 Conclusion We conclude that the CCLP method needs to be improved in order to be compatible with the other methods. Our studies also confirmed that measuring only bacterial amount is insufficient to determine caries risk; other factors need to be included to get a valid result. 4

5 5 Introduction Caries is the result of the interaction of many factors in the oral environment, for example the saliva flow and amount, presence of bacteria, enamel/tooth quality, plaque amount, oral hygiene, fluoride and sugar rich diets. It is one of our most common infectious diseases. Caries does not occur when there is equilibrium between attacking forces (sugar rich diets, bacteria) and the protecting forces (saliva, fluoride, good quality of teeth). Adverse variations of this equilibrium, for instance high sugar consumption and insufficient oral hygiene leads to a high amount of various caries bacterias, creating low ph and thereby destruction of the teeth (Reich E et al, 1993). Oral bacterias Streptococcus Mutans (SM) and Lactobacilli (LB) are well known for their role in the caries process. They have the ability to utilize sugars, produce acids, and adjust to a low ph environment. Even the bacterial species of S. Gordonii, S. Oralis, S. Mitis and S. Anginosus are capable of producing acids in a low ph environment as shown by Borgström (Borgström MK et al, 1997) and van Houte (Houte J Van, 1993). Sucrose is transported over the cell membrane into the bacteria via different transportation systems. The sucrose is metabolized via glycolysis that results in the production of energy for the life of the bacteria and lactic acid, which is a waste product. The lactic acid is transported out of the bacteria and lowers the ph in the surrounding plaque fluid and thus results in a critical ph drop that changes the equilibrium of the enamel crystals and leads to dissolution of the enamel crystals of the teeth (Linder L, Oral mirobiologi 60ff) Today prophylaxis is an important method to minimize caries incidents, but in order for the prophylaxis to be successful the patients in the risk group needs to be identified. There are several recognized methods for the identification of caries risk patients such as comparing the patients eating, drinking habits, oral hygiene with the (DMF t/s) index (decayed, missed, filled teeth or surfaces) (Reich E et al, 1993).

6 6 Another method is by detecting the amount of SM and LB in the patients mouth, where high bacteria levels indicate high caries risk (Carlsson J, 1989 and Cotter P, 2003). However, using only bacteria tests are not sufficient for detecting caries risk. Other factors such as eating habits, salivary flow, and oral hygiene should be involved (Fjerskow et al Clinical Dental Caries 54 f). These conventional methods (culturing of LB and SM) has been used for a long time to estimate bacteria count (Jensen B et al 1989), but it takes several days until the results of the tests are obtained. Another drawback was their high cost. Therefore, we wanted to evaluate a new, less costly, and a quicker test that could be used to screen all patients for high caries risk in order to give them proper programs to prevent caries development. The development of new, quick, methods for caries risk detection is desired. Clinpro Cario L-Pop (CCLP) is a new semi quantitative method that is manufactured by 3M ESPE, Seefeld, Germany. It is claimed by the manufacturer to detect caries risk patients in only a few minutes. The test is done by rolling the CCLP swab containing sucrose on the patient s tongue, and placed in the kits L-Pop blister. The lactic acid produced by the microorganisms on the tongue is monitored by a solution in the L - Pop blister that contains lactate dehydrogenase enzyme which facilitates the transformation of lactate in to pyruvate. This transformation is detected by the color indicator which makes the swab show a purple color within 2 minutes after the introduction of the enzyme (Häberlein et al 2003). The variation in the intensity of the purple color is graded from 1-9. The grade represents increasing amounts of acid produced and is presented in a chart where 1-3 represents low caries risk, 4-6 represents average risk and 7-9 represents high risk (see illustration one ). This method is dependent mainly on the acid produced by the oral bacteria capable of producing lactate. The method is an indicator of metabolic activity of caries-causing bacteria, and was adapted from earlier studies done during the seventies (Geddes DA, 1975). The idea of using lactic acid was based on the ecological plaque hypothesis; which is described as a disturbance in the dental biofilm which leads to a shift in the equilibrium. PH drop leads to acidogenic and aciduric bacterial benefits (Marsh PD,

7 7 1994). Lactic acids are a metabolic product from bacteria and may indicate the disturbance in the equilibrium. The mouth is harboring more than 700 bacterial species and only 40-50% can be cultivated (Fjerskow et al, Clinical dental Caries 178f), CCLP uses lactic acid as an indicator of adverse conditions (or shifts in the equilibrium) and might be a more sensitive method than the methods of counting bacteria using selective media. That is why it was interesting for us to test the CCLP and compare it with the other conventional methods. The experiments were performed in two different groups. One group consisted of dental students from Karolinska Institutet. The other group was recruited from visitors at the student dental clinic for adults or children and adolescents. The visitors were between years old. The dental status of both study groups was checked by dental students. The participants medical records, clinical status and oral hygiene habits were registered. Stimulated and none stimulated saliva flow were measured and saliva was collected for analysis. The saliva samples were sent to the laboratory for bacterial cultivation of SM and LB. The CCLP test was done and the results were compared to the other methods. Aim The aim of this study was to compare the CCLP test to the known conventional standard methods used to detect and quantify cariogenic bacteria as instructed at the Institute of Odontology, Karolinska Institutet, and to evaluate the reliability of this test. We have done a pre study to ascertain correct reading of the CCLP charts. Intra- and inter individual calibration tests were carried out using different concentrations of lactic acid to get different color intensity of the swab. The importance of the light conditions was tested by reading the swab color under different lamps and the results were compared.

8 8 Materials and Methods Calibration and validation of CCLP test Eighteen 4th year students were recruited from the dental program, to test the reading of the CCLP swab color and compare it with the reference color charts that belonged to the test kit. Three different concentrations of lactic acid, 0, 4 mm, 2 mm, 10 mm (Sigma Aldrich), were used in the calibration of CCLP color charts. The test swabs were dipped in the lactic acid and then replaced in their L- pop blisters, to allow lactic acid-enzymatic reactions to proceed. After 2 minutes, the swabs were removed and their colors were compared with the reference color chart of nine-fields. The results were interpreted by the students. Impact of light source on test interpretation The students were divided into two groups using two different light sources. The first light source was dental clinical light luminescent-screen tube Philips 2W/830 TL-5. The second light source was administrative office light low energy lamps 26W/ 830. Each student gave their own readings after comparing the color chart under daylight. The results were entered into a Microsoft Excel sheet and mean +/- SD and median was calculated for each lactic acid concentration. CCLP and other microbial tests Participants A total of 77 participants were recruited. One group is the students group and the other is a caries risk group (visitors). The visitor group consists of patients who visit the VUX clinic at the Department of Dental Medicine. The visitors were informed orally by us about the tests, information and procedures, and that they could withdraw at any time during the tests. They

9 9 agreed orally to participate in this study. In both groups we excluded those who had severe systemic diseases and those who had been using antibiotics during the last 3 months. The student group consisted of fifty-six volunteer dental students attending year two at the dental program at Karolinska Institutet. The caries risk group (visitors) consisted of twenty-one visitors with estimated to have high caries risk. The participants in the visitor group were selected according to the following criteria: Age above eighteen years, normal saliva flow rate, and high caries risk and caries active or high caries activity (at least 1-2 new caries lesions per year or more). Laboratory (lab) culturing of SM & LB Lab culturing of SM & LB and methods for execution of VMG: Stimulated whole saliva was collected for 5 minutes from both groups. The participants chewed a piece of paraffin wax, and the saliva flow was calculated (ml/min). Three ml of the stimulated saliva was added to four ml VMG II transport medium, shaken and then sent for lab selective culturing of SM and LB to the Department of microbiology at Karolinska. The saliva samples were diluted with a phosphate buffer solution to 10 4, 10 3, 10 2, 10 1 and one undiluted sample. Twenty µl of each solution were dripped on a Rogosa agar disk. Then twenty µl were dripped on a MSB (Mitis-sucrosebacitracin) disk. The disks were incubated anaerobically at 37 C for two days and the results were read. The results were given in bacteria collonieforming units (cfu) / ml saliva. (Frostell G, Oral mikrobiologi 18ff) (See box 1 and 2). Box 1 Laboratory results and interpretation of LB 1, cfu/ ml saliva Low 2, cfu/ ml saliva Medium 3, 100,000 cfu / ml saliva High

10 10 Box 2 Laboratory results and interpretation of SM 1, cfu/ml saliva Low 2, cfu/ml saliva Medium 3, cfu/ ml saliva High Dentocult LB Dentocult LB (manufactured by Orion Diagnostica, Finland) and methods of execution: Stimulated whole saliva was used to coat both sides of the Dentocult dipslide (covered with modified Rogosa medium). The dipslide was incubated, in an upright position, in an incubator at 37 O C for 4 days. The LB colonies were counted and registered according to the manufacturer s model chart. (See box 3 for reference values) Box 3 interpretation of Dentocult LB results 0, 0 cfu/ml Low caries risk 1, 1000 cfu/ml Low caries risk 2, cfu/ml Caries risk 3, cfu/ml High caries risk 4, cfu/ml High caries risk Clinpro Cario L-Pop (CCLP) Clinpro Cario L-Pop (manufactured by 3M ESPE Germany) and methods of execution: The participants in both groups were asked to brush their teeth with polishing paste RDA 40 (manufactured by CCS, Sweden). Five minutes after the tooth brushing was finished the CCLP tests were performed in accordance with the manufacture s guidelines. The readings were carried out using the color charts provided in the kit. The variation in the intensity of the purple color is graded from 1-9 and is presented in a chart where 1-3 show low caries risk, 4-6 average risk, 7-9 high risk. The color of the swabs was irregular distributed so the examiner was instructed to choose the darkest zone on the swab to compare with the chart to

11 11 interpret the result. The test was read by two different groups (clinical light and office light) during daylight and clinical light setting, (luminescentscreen tube Philips 2W/830 TL-5.) (See illustration 1 and box 4 for interpretation.) Illustration 1 the CCLP interpretation chart (Azrak B, 2008) Box 4 Interpretation of CCLP 1, Zone 1-3 in chart Low caries risk 2, Zone 4-6 in chart Average caries risk 3, Zone 7-9 in chart High caries risk Estimating caries risk: Using the following collected data (box 6 11); the caries risk was evaluated for each participant by comparing negative and positive factors together. The results were graded to low caries risk, medium and high (See box 5a, b for criteria.)

12 12 Box 5a caries risk 1, Low No negative factors 2, Medium A few negative factors, can potentially be improved by changing habits. 3, High Several negative factors, some risk factors cannot be changed. Box 5b Negative factors involved in caries risk assessment 1, Low fluoride exposure 2, Low saliva flow rate 3, High use of fermentable carbohydrates and intake frequency 4, Insufficient oral hygiene 5, Caries active 6, High caries frequency Collected anamnesis data Age, gender, medical history, medications, symptoms of oral dryness, diet and its composition (see box 6 for criteria s), smoking and snuff habits, fluoride exposure (see box 7 for criteria s) and tooth brushing habits se (box 8 for criteria s) Box 6, Use of fermentable carbohydrates and intake frequency 1, Low 5 food intakes / day, low general consumption of sugar 2, Medium 5 9 food intakes/ day, more frequent sugar consumption 3, High >9 food intakes / day, high frequent sugar consumption Box 7, Fluoride exposure 1, Low: toothpaste (>1000ppm) < 2 times a day 2, General recommendation: toothpaste (>1000ppm) 2 times a day 3, A bow general recommendation: toothpaste (>1000ppm) 2 times a day and extra fluorides e.g. rinse, pill

13 13 Box 8, Tooth brushing habits. 1, < Twice a day 2, Twice a day, (recommended base prophylaxis) 3 > Twice a day Clinical data Collection of stimulated (paraffin wax) and unstimulated whole salivary flow, using a local protocol established by the dental faculty of the Karolinska Institute. (See box 9a and 9b for reference values.) Box 9a, Unstimulated saliva flow: 1, 0, 25-0, 35 ml/min Normal 2, 0, 15 0, 25 ml/ min Low 3, 0-0, 15 ml/ min Very low Box 9b, Stimulated saliva flow 1, 1-3 ml/ min Normal 2, 0, 7-1 ml/min Low 3, 0 0, 7 ml/ min Very low Recording of cavitated teeth: Intra oral examination with probe and mirror to record initial and manifest cavities in a clinical unit setting. Bitewing x-rays were used to detect approximal lesions, not detectable visually, using Mejàre et al 1985 classification. (Fjerskov et al, Dental Caries p 72) By comparing new data with previous data collected in the participants dental chart, the caries activity (presence of active caries lesions) was evaluated. (See box 10.) To measure the c history of the participants we used the DMF-s index (sealed surfaces are regarded as not decayed and are not included.) (See box 11)

14 14 Box 10, Caries activity 1, Inactive No lesions at all 2, low activity No new lesions / year 3, Active A few lesions / year 4, High activity Several new lesions / year 5, Extreme high activity Severe new lesions / year Box 11, Caries frequency 1, <10 DMFs Very low 2, DMFs Low 3, DMFs Medium 4, DMFs High 5, 70 - DMFs Very high Statistical analysis Mean values, standard deviations and Pearson product-moment correlation coefficient (r), were calculated using Microsoft Excel and Prism GraphicPad software. In the correlation (r) statistics values < 0, 5 are regarded as no correlation. To interpret variations we merged the two study groups in to one and then divided the participants in to two groups. Group one: Caries inactive group no decayed surfaces (total caries free) 31 participants were included. Group two: caries active group, minimum one initial caries lesions, 46 participants were included.

15 15 Results: Comparison of caries risk assessment tests In the student group (n=56) the majority showed low results (low bacteria counts) with these tests: Dentocult LB 43 students (76%), Lab LB 46 students (82%), and Lab SM 54 students (96%). The Estimated caries risk correlated with the bacterial tests where the majority of students 46 (82%) showed low risk. In the student group the minority 7 (12, 5%) showed low results in the CCLP test, while the majority 32 (57%) showed high results. In these results we can see that the estimated risk in the student group is similar to the low levels of lab LB/SM, Dentocult LB, but not to CCLP. (Fig 1a) In the visitor group (n=21) the results of Dentocult LB, Lab LB, Lab SM were spread out between high, medium and low, while the majority in the student group showed low results with the bacterial tests. The estimated caries risk in the visitors` group showed that almost (50%) of this group had high risk and (50%) of this group had medium estimated caries risk, while in the student group the majority showed low estimated caries risk. CCLP results in the visitors` group showed also spread results, but the majority had high and medium results. These results clearly showed that the CCLP test gave different results compared to the bacterial tests. Also, the estimated risk was hard to compare, because the visitors group was a small study population (n = 21) and they were classified as caries active (Fig 1b).

16 Percentage of participants Percentage of participants % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Student Group (n=56) CC LP Dento LB Lab LB Lab SM Estim. Car Risk High Medium Low Figure 1a Dental student group: results on CCLP, Dentocult LB, Lab LB, Lab SM and estimated caries risk (n=56) Visitor Group (n=21) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CC LP Dento LB Lab LB Lab SM Estim. Car Risk High Medium Low Fig 1b Visitor group: results on CCLP, Dentocult LB, Lab LB, Lab SM and estimated caries risk (n = 21)

17 17 Comparison of Dentocult LB, Lab LB, Lab SM, CCLP, and estimated caries risk in both inactive and active participants groups Selection of caries active participant The student group and the visitor group were merged together and divided as following: forty percent of the participants showed no decayed surfaces (total caries free) (n=31), and were therefore named the caries inactive group. Sixty percent of the participants showed caries activity (n=46), thus named the caries active group, (minimum one initial caries lesion.) The criteria for choosing caries active participants were described previously in Materials and methods see box 10 (Fig 2). 40% caries inactive Caries active 60% Fig 2. Percentage of caries active and caries inactive (n= 77)

18 Percentage of participants 18 Caries inactive group: In the caries inactive group (n=31) the majority showed low bacterial counts with Dentocult LB, Lab LB, Lab SM. The bacterial test results correlated well with the estimated caries risk which also showed that the majority had low risk. However the CCLP results showed high and medium results for the majority. These results indicate that Dentocult LB, Lab LB, Lab SM, correlates to estimated risk results (Fig 3a). Caries active group In the caries active group (n=46) Dentocult LB, Lab LB, Lab SM have widespread results between low, medium, and high bacteria counts. The estimated caries risk in this group is also widespread. The CCLP results showed that the majority had medium and high results, and these results are similar to the caries inactive group (Fig 3b). 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Caries inactive (n=31) CCLP Dentocult LB Lab-LB Lab- SM Estim. Car Risk High Medium Low Fig 3a Caries inactive individuals and their results on CCLP, Dentocult LB, Lab LB, Lab SM and estimated caries risk (n = 31)

19 Percentage of participants % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CCLP Caries active (n=46) Dentocult LB Lab -LB Lab-SM Estim. Car Risk High Medium Low Fig 3b Caries active individuals and their results on CCLP, Dentocult LB, Lab LB, Lab SM and estimated caries risk (n = 46) Correlation studies The result obtained from the statistical correlation tests (r) shows that CCLP had no correlation to Dentocult LB (r = 0.2), lab LB (r = 0.05), lab SM (r = 0.05), and caries risk (r = 0.1). That can be due to the methodological difficulties that will be discussed later in this paper. Dentocult LB shows a good correlation to lab LB (r = 0.6), manifest caries lesions. (r =0, 7) and DMFs (r= 0, 5) a finding also reported by Nishikawara F et al, 2006). Lab SM shows good correlation to Initial caries lesion (r = 0, 7).

20 20 CCLP test color chart: The readings from the two different light sources Philips 2W/830 TL-5 and low energy lamps 26W/ 830 were compared. The results were entered into a Microsoft Excel sheet and mean +/- SD and median was calculated for each lactic acid concentration. Within each group there were no major differences as to the reading results. However, differences between readings under different lights were noticed, as seen in table 1. One issue noticed was the difference in the irregularity of the color of the swabs, which had created some difficulties in determining the color shade. This could be one reason underlying the variation between the observations (Table 1a and b). Tabel 1a Dental clinical light Lactic acid conc Tabel 1b Administrative office light Lactic acid conc Student 10 mm 2mM 0,4mM student 10mM 2mM 0,4mM Mean value 7,14 4,57 1, Median value 7,00 5,00 2,00 Standard deviation 0,38 1,27 0,69 Mean value 6,27 2,91 1,45 Median value 7,00 3,00 1,00 Standard deviation 1,85 1,04 0,69 Table 1a and b show the mean value, median value and the standard deviation of test groups in dental clinical and administrative office light.

21 21 Discussion All methods used singly for caries risk estimation could be criticized. However combining different methods provides better results in caries risk assessment. (Apostole P et al, 1986.) To be able to estimate the caries risk as much information as possible was collected. Therefore a protocol was made in which all the data collected from the participants, could be used to estimate the caries risk. Dental plaque formation on the teeth is a natural and common way of protecting the teeth from exogenous species. (Marsh PD 1994) However, homeostasis can break down, leading to changes in the balance of micro flora, thereby predisposing sites to disease, as written by Marsh PA (Marsh PD 1994). The consumption of carbohydrates, sugar, bad oral hygiene, lack of fluoride and the presence of specific bacteria like Streptococcus Mutans and Lactobacillus, are determining factors in caries development. The formation of lactic acid by the bacteria is the key factor of caries lesion development (Linder L, Oral mikrobiologi 60ff). That is why we wanted to test the new method Clinpro Cario L-Pop (CCLP) that measures the lactic acid production on the dorsum of the tongue, and compare it to other standard methods that detect the acid producing species SM, LB and can be used to estimate caries risk. The results we obtained from CCLP tests showed no correlation between Lab LB, Dentocult LB, Lab SM, caries activity and risk. The CCLP results were much different from the other results. There could be several explanations for these differences, one of them being that the CCLP method is not a selective bacterial test, and that other bacteria may be involved when performing the tests not only LB and SM species. Yet another explanation could be that the CCLP test is performed and collected from the dorsum of the tongue. It has in fact been shown that the anatomic differences on the tongue dorsum such as keratinized, pappillated surfaces with underlying serous glands create different microbiological environment and different ecology from other parts of the tongue and teeth (Aas J et al 2005). It has also been shown that the surface of the tongue serves as the initial reservoir for bacteria (Tanner ACR et al 2002).

22 22 One important explanation is related to the methodological difficulties of the CCLP method. Reading of the CCLP chart was complicated due to non homogeneity of the final color result on the swab, as also detected by another group (Azark B et al 2008). The color was not even and therefore we read the darkest color spots, and this may have caused over interpretation of the results. We found statistically significant correlation between lab SM and initial caries lesions, and between Dentocult LB and manifest caries lesions (0, 7). These findings can be compared with earlier studies that suggests that SM have a greater role in the initiation of dental caries and LB are associated with cavitated decays that needed to be restored. (Nishikawara F et al, 2006), (Fjerskov O et al Clinical dental Caries 179f). We also found that it is very important to have the same light source and surrounding conditions when performing the CCLP tests. It has been described by the manufacture that the swab should be read within 2 minutes after insertion to the lactate dehydrogenase enzyme. We have observed when performing our tests that any delay in the reading of the swab will lead to quick color changes within minutes. Therefore it is important to be consistent with the time taken to register the results. Any delay in taking the readings may result in over registration of the results. Also different results regarding the reproducibility of this test have been shown. One study showed reproducibility between 60-80% (Schiffner U et al, 2005), another showed 37% reproducibility (Gerardu V A M et al, 2006), and little information is known about the test. We calculated the percentage of non active (total caries free, no lesions) and active caries (minimum one initial) participant in order to use the figures in the analyses of our test methods. We found that the majority of non caries active and caries active participants showed low bacterial counts for Dentocult LB, lab LB, and lab SM. It is not surprising that non caries active participants have low bacterial counts, but what is surprising is that caries active participants have low bacterial counts. The conclusion can be that bacteria counts are not the only determining factor of caries activity, and that other parameters are involved in the determination of caries risk and

23 23 caries activity. This is also shown by others (Apostole P et al 1986). Another explanation of the low LB counts shown in caries active participants can be that Lactobacilli found in caries free patients and caries risk patients have different phenotype characteristics e.g. hydrophobicity and aggregation ability, and that can influence the results (Ahumad MC et al 2003). Many factors may be involved in caries risk predicators such as the diet, bacteria and the habits of the host. Previous tests have mainly focused on the bacterial aspect, and by only testing the bacteria insufficient results were obtained. That is why it is important to investigate as many factors as possible involved and to try to correlate them with each other. Conclusions Our conclusion is that CCLP did not give similar results to the other bacterial methods. We conclude that the CCLP test needs some improvement. We also found that it is difficult to predict caries risk by using any single method like Dentocult LB, lab LB or lab SM.

24 24 Acknowledgements We would like to thank our tutors Margaret Sällberg Chen and Anette Oliveby and 3M ESPE for providing the CCLP kits. As well as all the participants who volunteered in this study.

25 25 References Articles 1. Aas J.A, Paster B.J, Stokes L.N, Olsen I, Dewhirst F.E. Defining the normal bacterial flora of the oral cavity. Journal of clinical Microbiology, 2005 Nov: Ahumada MdC, Bru E, Colloca ME, López ME, Nader-Macías, Evaluation of lactobacilli characteristics in the mouths of patients whith or whitout cavaties, Journal of Oral Science vol 45/ 2003: Apostole P, Bacteriologic and nonbacteriologic criteria for identifying Individuals at high risk of developing Caries: A Review. Journal of public Health Dentistry vol 46 : Azark Birgül, Comparison of a new chairside test for caries risk assessment whith established methods in children, Schweiz Monatsshr Zhanmed vol 118 8/2008: Borgstrom MK, Sullivan A, Granath L, Nilsson G. On the ph lowering potential of lactobacilli and mutans streptococci from dental plaque related to the prevalence of caries. Community Dent Oral Epidemiol 1997; 25: Carlsson J. Microbial aspects of frequent intake of products with high sugar concentrations. Scand J Dent Res 1989; 97: Cotter P, Hill C. Surviving the acid test: responses of gram-positive bacteria to low ph. Microbiol Mol Biol Rev 2003; 67: Geddes DA. Acids produced by human dental plaque metabolism in situ. Caries Res 1975; 9:98-109). 9. Gerardu V A M, Heijnsbroek M, Buijs MJ, van der Weijden F, Cate JM ten, Loveren C Van. Comparison of clinpro Cario L-Pop estimates with CIA lactic acid estimates of the oral microflora. Eur J Oral Sci 2006; 114: Houte J Van. Microbiological predicators of caries risk. ADR 1993; 7: Häberlein, Kappler O, Schmid B, Guggen berger R. Measurment of lactic acid production of carious bacteria in the mouth: reability of the novel diagnostic device Clinpro Cario L- Pop.J Dent Res 2003; 82 Spec iss B, abs.no.2068: B-269.

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27 27 Handledarintyg Som handledare för detta projekt tillstyrker jag att studentens eller studenterna ska examineras eftersom dennes/deras prestation och insats i projektet och att den vetenskapliga rapporten är av tillräcklig omfattning och kvalitet för examination. [Datum] [NamnHandledare] Margaret Sällberg Chen Docent Anette Oliveby Docent