|DENTAL SCIENCE - ORIGINAL ARTICLE
|Year : 2013 | Volume
| Issue : 5 | Page : 5-9
A comparative evaluation of Oratest with the microbiological method of assessing caries activity in children
Meenakshi Sundaram1, Ullal Anand Nayak2, Krishnakumar Ramalingam3, Venugopal Reddy3, Arun Prasad Rao3, Mahesh Mathian4
1 Department of Pedodontics and Preventive Dentistry, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Rajah Muthiah Dental College and Hospital, Chidambaram, Tamil Nadu, India
3 Department of Peadiatric and Preventive Dentistry, Mahatma Gandhi Dental College and Hospital, Jaipur, Rajasthan, India
4 Department of Peadiatric and Preventive Dentistry, Vivekanandha Dental College for Women, Tiruchengode, Tamil Nadu, India
|Date of Submission||02-May-2013|
|Date of Decision||04-May-2013|
|Date of Acceptance||04-May-2013|
|Date of Web Publication||13-Jun-2013|
Department of Pedodontics and Preventive Dentistry, Vishnu Dental College, Bhimavaram, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims: The aim of this study is to find out whether Oratest can be used as a diagnostic tool in assessing the caries activity by evaluating its relationship to the existing caries status and the salivary streptococcus mutans level. Materials and Methods: The study sample consists of 90 students divided into two groups. Group I (test group) and Group II (control group) consisting of 30 children for control group and 60 children for test group. The sampling of unstimulated saliva for the estimation of streptococcus mutans was done as per the method suggested by Kohler and Bratthall. The plates were then incubated. Rough surface colonies were identified as streptococcus mutans on a pre-determined area of the tip (approximately 1.5 cm 2 ) were counted for each side of spatula pressed against mitis salivarius bacitracin agar using digital colony counter. The results were expressed in colony forming units (CFU). Oratest was carried out in the same patients after the collection of salivary sample for the microbiological method to evaluate the relationship between the two tests. Statistical Analysis Used: The tests used were ANOVA, Pearson Chi-square test, Pearson's correlation analysis, Mann-Whitney U test and Student's independent t-test. Results: In the control group and test group, when the streptococcus mutans count (CFU) and Oratest time (minutes) were correlated using Pearson's correlation analysis, the streptococcus mutans counts was found to be in a statistically significant negative linear relationship with the Oratest time. When the caries status of the children, participated in the test group were correlated with mutans count (CFU) and Oratest time, caries status were found to be in a statistically significant positive linear relationship with streptococcus mutans count and in a significant negative linear relationship with Oratest time. Conclusions: The test proved to be a simple, inexpensive and rapid technique for assessing caries activity since a significant relationship exists clinically with caries status and microbiologically with the streptococcus mutans count of the individual.
Keywords: Caries activity, Oratest, streptococcus mutans
|How to cite this article:|
Sundaram M, Nayak UA, Ramalingam K, Reddy V, Rao AP, Mathian M. A comparative evaluation of Oratest with the microbiological method of assessing caries activity in children. J Pharm Bioall Sci 2013;5, Suppl S1:5-9
|How to cite this URL:|
Sundaram M, Nayak UA, Ramalingam K, Reddy V, Rao AP, Mathian M. A comparative evaluation of Oratest with the microbiological method of assessing caries activity in children. J Pharm Bioall Sci [serial online] 2013 [cited 2022 Nov 28];5, Suppl S1:5-9. Available from: https://www.jpbsonline.org/text.asp?2013/5/5/5/113283
Dental caries is a common infectious disease worldwide. Mutans streptococci are the main etiological microorganisms in dental caries.  A quantitative measure of streptococcus is said to be a reliable measure to predict true caries activity.  A significant correlation exists between the number of mutans streptococci in saliva and their prevalence in dentition. Salivary tests can elucidate the true risk. 
Dental caries activity tests have been widely used in the assessment, monitoring and motivation of patients with dental caries. Microbiological caries activity tests are chiefly based on two principles,
The quantification of mutans streptococcus using mitis salivarius bacitracin (MSB) agar by conventional microbiological method is well documented in the literature.  However, in routine clinical practice, these caries activity tests require specially prepared culture media and laboratory facilities for incubation or expensive kits to perform them. 
- Estimation of bacterial counts expressed as colony forming units (CFU).
- Qualitative or quantitative estimation of products of bacterial metabolism. 
A chair-side simple, economical, non-invasive method used for the detection and enumeration of oral microbial level based on the rate of oxygen depletion in expectorated milk samples, called "Oratest", was developed by Rosenberg, et al. in 1989.  They reported that the time taken for the indicator dye methylene blue in the milk expectorated after rinsing of the mouth was inversely proportional to the number of organisms in the expectorate.
Subsequently, they studied Oratest in gingivitis, halitosis and also as a method to monitor denture hygiene. Later, in 1996 Patalay, et al. attempted to use Oratest as a method to assess caries activity since dental caries has been currently accepted to be a specific microbial disease. 
Although both streptococci mutans count and the Oratest seem to be promising for caries risk assessment, comparisons between these two methods have been scarcely done. Till date, no studies are so far reported. In view of the racial difference in the dietary habits, composition of the teeth and the microbiota harboring the oral cavity, the study of a new caries activity test, which is simple, was done using Oratest with the following aims and objectives:
Principle of Oratest
- To find the correlation between the Oratest and the streptococcus mutans counts in the salivary samples in children.
- To find the correlation between the existing caries status and salivary streptococcus mutans.
- To find the correlation between the existing caries status and the Oratest.
Oratest is based on the rate of Oxygen depletion by microorganisms. Bacterial enzyme (aerobic dehydrogenase) transfers electron or proton to oxygen. Once aerobic organisms utilize oxygen, aerobic dehydrogenase transfer of electrons from substrate milk reduces methylene blue to leucomethylene blue. This shows the metabolic activity of aerobic organisms. 
Oratest is based on whole mouth rinsing with sterile milk. Milk is a suitable liquid for dislodging microorganisms and provides an excellent medium for subsequent metabolism of microorganisms. As milk is white in color it is easier to detect the earliest color change from blue to white at the base of the test tube. 
| Materials and Methods|| |
Out of 512, 6-12 years aged school going children of Chidambaram, 90 children (49 boys and 41 girls) were selected. This study was carried out after obtaining the parent consent. The study sample was divided into two groups. Group I (Test group) and Group II (control group) consisting of 30 children for control group and 60 children for test group. The criteria for selection of subjects in Group I was, children with dental caries involving one or more teeth and Gingival index score of Zero (Loe and Silness index). Group I were equally divided into 1, 2 and 3 sub-groups according to the deft and decayed missing filled teeth (DMFT) of 2-5, 6-10 and 11-18 respectively. The criteria for selection of subjects in Group II was, children free from caries, gingivitis and plaques score of zero as per modified Silness and Loe plaque index.
Microbiological method was performed in the patients earlier to Oratest. Before dental examination, media used for microbiological study was prepared. Media used for culturing the microorganisms (mutans streptococci) in the present study is MSB agar (Gold, et al. 1973). 
Composition of media
Constituents of MSB agar per 1 l in
- Mitis Salivarius dehydrated agar - Himedia company.
- Potassium tellurite - 0.1 mg/ml.
- Sucrose - 20% W/V.
- Bacitracin - 0.2 units/ml.
- Distilled water - 1 l [Figure 1].
Collection of sample for microbiological evaluation
The sampling of unstimulated saliva for the estimation of streptococcus mutans was done as per the method suggested by Kohler and Bratthall in 1979.  3 cm of the sterilized wooden spatula was introduced into the mouth and turned around ten times to contaminate with saliva. Excess was wiped against the lips while taken out of the mouth. Each spatula was then stamped with same force in the centre of the plate, with this spatula method, grown colonies were restricted. 
The plates were then incubated within 4 h at 37°C for 48 h under 5-20% CO 2 . Round or spherical, highly convex, raised, dark blue in color, ranging from a pinpoint to pin head size with a rough surface colonies were identified as streptococcus mutans on a pre-determined area of the tip (approximately 1.5 cm 2 ) were counted for each side of spatula pressed against MSB agar using digital colony counter. The results were expressed in CFU.  To avoid bias, all plates were processed and examined by the same investigator. Confirmation of mutans streptococci was performed under light microscope.
Oratest was carried out in the same patients after the collection of salivary sample for the microbiological method to evaluate the relationship between the two tests.
The armamentariums used were:
- Sterile beakers - Screw cap test tubes.
- Sterilized milk - 0.1% methylene blue solution.
- 10 ml disposable syringes - Sterile pipettes.
- Mirror-test tube stand [Figure 2].
Each patient was given 10 ml of ultra-high temperature sterilized cow milk and asked to rinse the mouth vigorously for 30 s. The expectorate was collected in the same beaker and 3 ml of this was transferred to screw cap test tube with the help of a disposable syringe. To this 0.12 ml of 0.1% methylene blue (0.1% methylene blue was obtained by mixing 100 mg of methylene blue in 100 ml of distilled water.) was added, thoroughly mixed and then placed on a stand in a well-illuminated area over a mouth mirror at room temperature. Test tubes were observed every 10 min for color change at the bottom, which would easily be discernible in the mirror. Time taken for initial color change within a 6 mm diameter circle on the bottom of the test tube was recorded.
The tests used were ANOVA, Pearson Chi-square test, Pearson's correlation analysis, Mann-Whitney U test and Student's independent t-test.
| Observations and Results|| |
The study was carried out in ninety children with 2-5 DMFT, 6-10 DMFT and 11-18 DMFT, and they were grouped as test groups with subgroups 1, 2, 3 respectively and caries free were grouped as control group based on their oral findings.
In the Oratest test, the time taken for color change in children with no caries, ranged from 247 min to 305 min with a mean of 271.4 ± 14.4 min, whereas, in the test groups i.e., caries group ranged from 22 min to 200 min with a mean of 84.1 ± 59.4 min. however, the Oratest time in control group was significantly higher than test groups ( P < 0.0001) [Table 1].
The mean time taken for color change in the Oratest in 2-5 DMFT (Test I group), 6-10 DMFT (Test II group) and 11-18 DMFT (Test III group) groups were 164.9 ± 18.5 min, 53.6 ± 6.8 min, 33.8 ± 6.4 min respectively. Time taken for color change is inverse of mutans level. Hence, it shows a negative linear correlation between Oratest and count on Pearson correlation coefficient analysis. Comparison of Oratest time between three test subgroups was significant ( P < 0.0001) using one-way ANOVA [Table 2].
|Table 2: Mean, standard deviation and test of significance of mean values between different study groups|
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Streptococcus mutans count in children with no caries ranged from 4 CFU to 30 CFU with a mean of 22.3 ± 7.3 CFU, whereas, in the test groups i.e., caries group ranged from 47 CFU to 174 CFU with a mean of 106 ± 42.1 CFU. however, the mean number of count in test group (106 ± 42.1) was significantly higher than control group (22.3 ± 7.3) ( P < 0.0001) [Table 1].
The mean streptococcus mutans level of test groups i.e., 2-5 DMFT (Test I), 6-10 DMFT (Test II), 11-18 DMFT (Test III), groups were 56.4 ± 10.1 CFU, 109.6 ± 12.1 CFU, 154.9 ± 11.9 CFU respectively. In the test group, 11-18 DMFT group showed higher level than the rest. Comparison of mutans levels between three test subgroups was statistically significant ( P < 0.001) using ANOVA [Table 2].
In the control group and test group, when the streptococcus mutans count (CFU) and Oratest time (minutes) were correlated using Pearson's correlation analysis, the streptococcus mutans counts was found to be in a statistically significant negative linear relationship with the Oratest time (r = −0.081, P < 0.0001) (r = −0.093, P < 0.0001) [Table 3]. When the caries status of the children, participated in the test group were correlated with mutans count (CFU) and Oratest time, caries status were found to be in a statistically significant positive linear relationship with streptococcus mutans count (r = 0.98, P < 0.0001) and in a significant negative linear relationship with Oratest time (r = 0.098, P < 0.0001) [Table 4]. In the present study, P < 0.05 was considered as the level of significance.
| Discussion|| |
Dental caries activity tests have been widely used in the assessment, monitoring and motivation of patients with dental caries and still caries activity test is under the continuous challenge due to its multi-factorial nature.  However, caries is now considered to be a specific odontopathic infection, the principal causative organism being mutans streptococci.  Streptococcus mutans is acidogenic and aciduric and once established, it can survive even in an unfavorable condition.  the positive correlation between streptococcus mutans and dental caries is well established. 
The growing interest in the microbiological aspect of dental caries has led to the development of a variety of diagnostic approaches. Some investigators have suggested relationships between the presence streptococcus mutans and evidence of dental caries and others proposed to detect microbial products to measure the caries activity. Unfortunately, many of these approaches require extensive work-up time and additional equipment, including incubators and microscopes for morphological count, often samples must be sent out for analysis. A simple in-expensive technique that does not demand sophisticated skills or valuable costly "chair-side" time, required in order to expedite the diagnosis and appropriate management of dental caries.
Mitis salivarius (MS) agar is a selective medium for culturing the four strains of streptococci species namely mitis, salivarius, mutans and angionus. By addition of 0.2 units/ml bacitracin and 20% sucrose to MS led to an improved medium (MSB) with high selection of streptococcus mutans. 20% sucrose causes mutans to form colonies with characteristic morphology and bacitracin to suppress the growth of other species like S. salivarius isolation for caries related streptococcus.  Selectivity of MS was further improved by the supplementation with 1% potassium tellurite.
In the present study with Oratest, mean time taken for color change from blue to white was 271 ± 14.4 min in the control group, whereas, in the test group it was 84.1 ± 59.4 min. Mean time taken for color change Test I, II and III group were 164.9 ± 18.5 min, 53.6 ± 6.8 min and 33.8 ± 6.4 min respectively. These findings were almost similar to the result of Patalay et al. (1996). 
Microbiological count of Streptococcus mutans using MSB agar from unstimulated saliva, in the control group was 22.3 ± 7.3 CFU, whereas, in the test group it was 106 ± 42.1 CFU. Mean CFU in the Test I, II and III group were 56.4 ± 10.1, 109 ± 12.1 and 54.9 ± 11.9 respectively. These findings were almost similar to the results of Ali, et al. (1998). 
A definite relationship exists between CFU of streptococcus mutans (microbial factor), caries status in both control group and test group (clinical factor) and Oratest time (caries activity). These findings were similar to the result of Munshi, et al. (1999),  Ali, et al. (1998)  and Leonor (2001). 
This study suggests the Oratest as a diagnostic tool to assess the caries activity since a definite relationship exist clinically with caries status and microbiologically with the streptococcus mutans count of the individual. This present study describes a new method for the enumeration of mutans streptococci in saliva. We believe that this method will be useful, both at the individual level and community level, for example, when evaluating important caries risk factors in a single case or monitoring a patient undergoing preventive treatment and at a population level, as one measure, in an attempt to identify groups of persons with an increased risk to develop caries.
| Conclusion|| |
Oratest is proven to be a simple, inexpensive and a rapid technique for the enumeration of mutans streptococci in saliva. We believe that this method will be useful, both at the individual level and community level, when evaluating important caries risk factors in a single case or monitoring a patient undergoing preventive treatment and at a population level, as one measure, in an attempt to identify groups of persons with an increased risk to develop caries.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
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