Oratest: Old Wine In New Bottle!

INTRODUCTION

The oral cavity is inhabitated by a plethora of microorganisms involved in various diseases of which dental caries is of special concern to mankind¹. Carious lesions can be easily detected by a clinical examination with a mouth mirror and explorer coupled with bitewing X rays. However, a clinical examination neither predicts caries activity nor indicates a patient's susceptibility to dental caries² .

Since years altogether, the use of caries activity tests is widespread in the field of dental research and clinical dentistry. Most of these caries activity tests are based on the concept of specific odontogenic infection, the principle causative organism being Streptococcus mutans. Their predominence is attributed to acidogenic and aciduric nature after a selective growth advantages over the other non acid tolerant organisms³ .Many studies on caries activity are aimed at finding relevant micro organisms. But to date, the ideal method to evaluate in terms of sensitivity, specialization and reliability has not been found. However, in routine clinical practice these caries activity tests require extensive armamentarium like specially prepared culture media, laboratory facilities for incubation and expensive kits to perform them⁴.

A reliable, inexpensive, chairside, noninvasive method for estimation of oral microbial levels termed as Oratest was developed by Rosenberg and coworkers in 1989⁵ . In this test the subjects rinse their mouth with sterile milk which dislodges the micro-organisms and also produces a substrate for their further metabolism. The expectorate is then added to methylene blue and color changes are observed over a period of time. Oratest is based on the rate of oxygen depletion by micro organisms. Under aerobic conditions the bacterial enzyme, aerobic dehydrogenase transfers electrons or protons to oxygen. Once oxygen gets utilized by the aerobic organisms and an anaerobic environment is attained, methylene blue [redox indicator] acts as an electron acceptor and gets reduced to leucomethylene blue. The metabolic activity of the aerobic microorganism is reflected by the reduction of methylene blue to leucomethylene blue. The formation of leucomethylene blue can be easily observed because of the white color of milk^5,6

Validation of any technique/test requires more studies to be done. As there is limited literature available regarding Oratest, the present study was carried out with the following

OBJECTIVES

1. To evaluate relationship of plaque index with Oratest

2. To evaluate relationship of caries status with Oratest

3. To compare plaque index scores and caries status.

MATERIALS & METHODS

The study was approved by Institutional Ethics Committee and informed consent was obtained from parents prior to initiation of the study. 50 healthy children (mean age 7 to 12 yrs) were randomly selected from government school in Belgaum city. All subjects were recruited only after a lapse of approximately 90 min since their last consumption of food or drink.

The study sample was divided into 2 groups: Group I (experimental group) consisting of 25 children with dental caries and group II (control group) with 25 children without dental caries. Criteria for selection of experimental group were dental caries involving more than one or more teeth clinically and radiographically, absence of abscess, draining sinus or cellulitis clinically. Criteria for selection of control group were clinical and radiographic absence of caries. The experimental group was divided into 3 subgroups according to the dmft/DMFT values which are showed in the table below:

The armamentarium used were ultra high temperature sterilized cow's milk, 0.1% aqueous solution of methylene blue, sterile beakers, screw cap test tubes, 5ml disposable syringes, sterile pipettes, mirror, test tube stand, digital stop watch.

For Group I, dmft/DMFT scores were calculated and the higher value was noted. Plaque scores for both Groups were calculated using TureskyGilmore-Glickman modification of Quigley-Hein index. 

Procedure

Each child was asked to rinse the mouth vigorously for 30 seconds with 10 ml of ultra-high temperature sterilized cow's milk containing 3% of fat. [Figure 1] The expectorate was collected in the sterile beaker. Using a disposable syringe, 3 ml of this was immediately transferred to a screw cap test tube which already contained 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.} The expectorated milk and methylene blue was thoroughly mixed and the test tube was placed on a stand in a well illuminated area. [Figure 2] A mirror was placed at the bottom of the test tube to detect any color change [blue to white] every 10 minutes. [Figure 3] The time taken for the initiation of the color change within a 6 mm ring of the bottom of the test tube was recorded by a second investigator who was blinded to the procedure of selection of groups.[Figure 4] A digital calibrated stopwatch was used to record the time.

The data was fed into the computer with Excel and statistical analysis was done. The tests used were unpaired t test, Mann-Whitney “U” test, Pearson's correlation coefficient and Spearman's rank correlation coefficient method.

RESULTS

The study was conducted in a total of 50 children and were divided into experimental group (group I) and control group (group II) of 25 children each. The mean time for color change for control group was 150.36 + 21 min and for experimental group was 85.60 + 5 min. Comparison of both the groups was done by unpaired t test and was found to be statistically significant (P=0.0046*) [Table 1]. 

The time for color change observed in subgroup A witha mean dmft/DMFT of 2.41 and 12 subjects, was 104.25 min, for subgroup B with a mean dmft/DMFT of 4.67 and 6 subjects it was 47.8 min while for subgroup C with mean dmft/DMFT 7.8 and 7 subjects it was 42.7 min. The correlation between the dmft/DMFT values for total samples and time taken for colour change was done using the Pearson's correlation coefficient method and Spearman's rank correlation coefficient method. The Pearson's correlation coefficient value (r) was - 0.7511; P=0.000* and Spearman R-value was - 0.7050, P=0.0001*. The Pearson's correlation coefficient value (r) for subgroup A, B and C were 0.7564; P=0.0044*, -0.2048; P=0.6970 and - 0.0929; P=0.8430 respectively. The Spearman R-value forsubgroup A, B and C were 0.7399; P=0.0059*, 0.0626; P=0.9062 and 0.0000; P=1.0000 respectively [Table 2 and 3].

The correlation of plaque index scores with time taken for color change was done by Pearson's correlation coefficient method and Spearman's rank correlation coefficient method. The Pearson's correlation coefficient value (r) for for group I was - 0.1501; P=0.4739 and for group II was -0.5540; P=0.0041*. The Spearman R-value for group I was -0.1765; P=0.3988 and group II was -0.5823; P=0.0023* [Table 4 and 5]. Comparison of both the groups for plaque index scores was done by MannWhitney U test. Group I had mean value of 1.32 + 0.18 and group II had 1.25 + 0.26 and their comparison was not found to be statistically significant (P=0.2405) [Table 6].

Also multiple regression of time with dmft/DMFT and plaque index was done with regression coefficient being -6.2253; P=0.0010* and -29.5599; P=0.2081 respectively [Table 7]. Both the dmft/DMFT and plaque index scores showed negative correlation.

DISCUSSION

The unique and ubiquitous nature of dental caries has led to it being the oldest and most common disease of mankind. The growing interest in the microbiological aspect of dental caries has given birth to the development of a variety of diagnostic approaches³. The clinical examination and quantification of factors associated with the pathogenesis of caries like the host, micro flora and diet is the key to an objective evaluation of caries activity. The use of dental caries activity tests in assessment of these factors has crossed leaps and bounds. They serve as an important tool in establishing the risk for caries and also for imparting preventive measures to all subjects at risk. Although literature demonstrates numerous tests, none of the currently available methods are completely satisfactory and reliable⁷. Many of these tests require extensive work-up time and additional equipment like incubators and microscopes for morphological count, often samples must be sent out for analysis. The reliability of such tests is limited mainly for two reasons. Firstly many of these tests rely on the samples of salivary bacteria and secondly the bacteria that are free-floating in the saliva may not necessarily represent the bacteria in plaque. A simple, cost effective technique overcoming the above limitations and which requires minimally skilled personnel is needed³.

In the present study 50 healthy school going children were randomly selected. The test was based on whole mouth rinsing with sterile milk. Milk is a suitable vehicle which dislodges microorganisms mildly yet effectively. It is nontoxic, provides an excellent medium for subsequent metabolism and also readily acceptable by the children⁸ . The expectorate and methylene blue in the test tube were thoroughly mixed and the time taken for initiation of color change was noted. The time taken for color change of methylene blue was compared with the deft/DMFT values and plaque index scores.

For the experimental group, the time taken for color change was 85.60 min ± 5 and 150.36 min ± 21 for the control group. Comparison between the means of both the groups was found to be highly significant (P=0.0046*). Amongst the three subgroups of experimental group, maximum time for colour change was found with subgroup A(with lowest dmft/DMFT value) and least with subgroup C (with highest dmft/DMFT value). The correlation between the dmft/DMFT and time was found to be negatively correlated for subgroup B and C. These findings were in agreement with the findings of Arora et al. who performed Oratest on 60 children. They found the mean time taken for the color change was 145-190 min in the control group and 90-130 min for the experimental group. The difference between the 2 groups was highly significant⁸. Thus as dmft/DMFT values increased Oratest scores decreased and vice versa. However subgroup A showed a positive correlation between dmft/DMFT and time. The suitable explanation for the difference in correlation between the subgroups with the time taken could be because of the different number of subjects in each subgroup. Had there been equal number of children in each subgroup then the results would have been similar.

For calculating plaque index scores, there were 2 main reasons for using Turesky-GilmoreGlickman modification of Quigley-Hein index. Firstly it is recognized as a reliable index for measuring plaque using an estimate of area of tooth covered by plaque; secondly it is relatively easy to use because of objective definitions of each numerical score. In the present study when plaque scores of both the groups were compared, no significant correlation was found between the scores and time taken for color change. The possible justification for this would be the mean values lying in the range of 1 to 2 for both the groups. However when plaque scores were correlated with time, the values of both the groups were negatively correlated. Tal Haim and Rosenberg Mel observed similar results when they compared Oratest scores with commonly used techniques for clinical evaluation of plaque levels and gingival inflammation. They also reported that higher the Oratestscores, lower the value of plaque index. In addition to this, it was found that children with increased scores of plaque index were within the age group of 11 to 12 yrs in control group and 7 to 8 yrs in experimental group. Thus as age increased scores of plaque index also increased and oratest scores decreased. These findings were in agreement with those of Joshi M et al. who evaluated whether Oratest could be used as a sensitive indicator of plaque levels and gingivitis⁹. This can be explained by the fact that the association between mutans streptococci and dental caries is not unique although they are considered to be crucial in development of caries. Thus caries can occur in apparent absence of streptococci while these species can prevail even without evidence of detectable demineralization. In such situations some acidogenicnonmutans streptococci are responsible for the disease¹⁰.

These facts comply with the findings of the study by Marsh PD who comprehended the glycolytic activity of oral streptococci. He found that some strains of nonmutans streptococci (e.g. Streptococcus mitis biovar 1 and Streptococcus oralis) can still metabolise sugars to acids at a mode r a t e ly low ph. The rate at which matabolisation occurs is comparable to that achieved by mutans streptococci¹¹.

Thus caries prevention strategies should include that of reduction of acid production and maintenance of plaque pH around neutrality. The present study, therefore, proved that higher the dmft/DMFT and plaque index scores, lesser was the time taken for the change in color of the expectorate.

CONCLUSION

In clinical management of children, it is difficult to identify them as caries susceptible solely on the basis of visual and tactile methods of examination. To avert this crisis caries activity tests come into picture because they can determine the extent of need of personalized preventive measures. Thus oratest, meets the need of an ideal caries activity test along with no necessity of any sophisticated skills to perform. It can therefore serve as an index for success of preventive and therapeutic measures and a valuable tool for education of preventive dentistry in school dental health programmes. Its role in pediatric dentistry is hence, unprecedented.

Limitation of the study

Firstly the sample size was too small. Secondly in the present study the sample was first selected and then divided in various groups (dmft / DMFT). However if samples were selected on the basis of dmft / DMFT scores then the results could have been more specific and interpreted accurately.