Detection of Oral Microbial Levels with Chair Side and Laboratory Method in Children with Caries: A Randomized Double-blind Controlled Study

INTRODUCTION

Throughout the world, dental caries is among the most prevalent infectious diseases.¹ When one or more decayed (cavitated or non-cavitated) lesions, missing (cavitated), or filled tooth surfaces occur in any primary tooth of a child 71 months of age or younger, it is referred to as early childhood caries (ECC).²

Managing ECC requires developing both restorative and preventive strategies. Since caries is a multifactorial disease, predicting caries risk and the effectiveness of preventive measures depends on numerous factors, including the microbial count.

Since Streptococcus mutans (S. mutans) is the primary causative agent of dental caries, a quantitative measurement of S. mutans is considered a reliable indicator of actual caries activity.³ Various techniques, both traditional and contemporary, are employed to measure S. mutans counts. The traditional method, considered the “gold standard,” involves culturing S. mutans from saliva samples in a laboratory to count the colony-forming units. The preferred medium for this culture is Mitis-Salivarius-Bacitracin (MSB) agar, as established by Gold et al. in 1973.³

The Oratest is a microbiological assay that assesses aerobic microbe activity by measuring the rate at which methylene blue in a milk substrate changes from red to leucomethylene blue. Aerobic microbes use oxygen through the process of oxygen-mediated electron transfer, or aerobic dehydrogenase.

A well-equipped laboratory and at least 48 hours of incubation are needed for the culture test to identify detectable amounts of S. mutans. In contrast, chairside strip tests, used to measure the number of colony-forming units (CFU) of S. mutans, have been available in various forms for some time. A new rapid chairside method, the Saliva-Check Mutans immunoassay system, utilizes a monoclonal antibody system where detection is based on antigen-antibody reactions. This system is designed to provide highly specific and immediate results within 15 minutes, offering a quicker and more convenient approach for S. mutans detection that is both fast and easy to perform.⁴

The present study was designed to compare and correlate the efficacy of three salivary microbial detection tests, i.e., laboratory S. mutans culture test, Oratest and Global Corporation (GC) Saliva Check Mutans kit test, in children with and without ECC. The goal of the research is to assess the efficacy of chair side methods as an effective motivational and educational tool for patients. The aim of the study is to find the correlation of the tests for S. mutans with the caries status of the children. The objectives are (a) To enumerate the S. mutans count in children with and without ECC by laboratory S. mutans culture and the GC Saliva Check Mutans kit test, (b) to detect the level of aerobic microbial activity in the saliva through Oratest in the same children.

MATERIALS AND METHODS

Ethical clearance for the study was granted by the institute, and all the Institutional Ethical Committee’s and the Central Ethics Committee on Human Research’s (India) ethical criteria were followed.⁵ The study was conducted in the city of Modinagar, for a period of 1 month. The study was designed as a randomized double-blind controlled trial (Fig. 1) and was conducted on children under 71 months of age who visited the Out Patient Department (OPD) of the Department of Pediatric and Preventive Dentistry at the Institute of Dental Studies and Technologies, Modinagar. All children with caries were examined by one single examiner using visual and tactile methods. Intra-oral examination was done using a mouth mirror, and an explorer. DEFT scores were recorded as per WHO criteria. Gingival index was noted for each patient. The children with a maximum age of 71 months with no caries and equal to more than 5 carious lesions who were able to spit saliva in the vials were selected. To include the children in the study it was required that they should not have a medical history or a history of long-term drug use, have a gingival index score of zero, and show no signs of abscess, draining sinus, or cellulitis in relation to any tooth.

RESULTS

Both the groups were subjected to Chi-square test and Pearson’s correlation analysis.

DISCUSSION

The evaluation of all risk variables enables a more precise prediction of the development of caries, which makes caries risk assessment crucial. It aids in estimating the chance of caries occurrence within a specific time frame and in comprehending the likelihood that the lesion would alter in size or activity.⁷

Many caries prediction models often include the salivary levels of S. mutans.⁸ Klock and Krasse originally reported on the quantitative study of S. mutans in saliva, and they discovered a direct correlation between the microorganisms’ concentration in saliva and the presence of bacterial plaque on tooth surfaces. For dental plaque to be representative of the entire mouth, it must be gathered from numerous teeth. Saliva is therefore a better diagnostic tool for measuring the S. mutans count because it is easier to sample.⁹ Additionally, it is simple to gather and includes systemically and locally generated markers of oral illness.¹⁰

In the present study, stimulated saliva was used to determine the S. mutans count, as stimulation of saliva results in a flushing effect and the clearance of oral debris and noxious agents.¹¹

A high caries rate has been associated with an S. mutans count of 5 × 10⁵ by Leal SC and Mickenautsch, who have summed up this conclusion of high levels of S. mutans after reviewing and collecting the available data.¹² In the present study, a mean S. mutans count was seen to be 6.39 × 10⁵ CFU with a mean DEFT score of 7.

Multiple research have evaluated the predictive usefulness of salivary levels in S. mutans, with inconsistent findings. There is a noteworthy correlation between salivary levels of mutans streptococci and the eventual onset of caries, as reported by some researchers; however, other investigations did not find this correlation.¹³ These detected differences can be attributed to the different methods of saliva collection, caries status and methods employed in estimating the S. mutans count.

When salivary S. mutans were cultured with MSB agar, it was found that with an increase in the caries rate, the levels of S. mutans also increased.

A positive outcome of the test is present only when the S. mutans count is above 5 × 10⁵ or it can be stated that only when the DMFT/DEFT score is quite high, GC test will be positive. In the present study, 8 out of 10 children in the study group showed positive results. The culture test of the two participants who tested negative in the study group showed an S. mutans count of 4.7 × 10⁵ and 4.6 × 10⁵ CFU. Thus, the present result reveals the highly sensitive and specific nature of this test in identifying the exact number of S. mutans. This kit uses a highly specific immunochromatography technique to identify S. mutans in saliva. Salivary S. mutans reacts with the anti-S. mutans monoclonal antibody tagged with colloidal gold on the test equipment. As a result, S. mutans’ surface is coated in gold colloidal particles. This creates the red line on the test window when it combines with another S. mutans antibody.

A total of 190 children ages from 3 to 4 were subjected to a chairside test comparison by Gao XL et al. They discovered that the GC Saliva Check Mutans, with a sensitivity of 97.6% and specificity of 90.6%, was more useful and reliable.⁴ Twetman L and Twetman S examined the efficacy of Dentocult SM and GC Saliva Check Mutans in 89 persons aged 23–72 years, and discovered that both were efficient for chairside detection of saliva check mutans, with a sensitivity of 80% and a specificity of 88%.¹⁴

Oratest was initially created by Tal H and Rosenberg M as a, chairside, non-invasive technique for determining the oral cavity’s bacterial population.¹⁵

According to an Oratest conducted by Bhasin S et al., the control group took 148 ± 38 minutes, while the mean duration for color change was 36 ± 8 minutes for 24 children with a DMFT of 6–11.⁶ According to Saxena S et al., color change for 50 children with a DMFT of ≥1 took 55.6 ± 2.31 minutes, but the comparison group, with a DMFT of 0.16, took 278.5 ± 1.50 minutes.¹⁶

In a study with 512 children, Sunadaram M et al. found that the time it took for an Oratest to test positive was 53.6 ± 6.8 and 271.4 ± 14.4 minutes in the test group (DMFT of 6–10) and the control group (DMFT of 0), respectively.³ Chandak S et al. found that the time it took for a color change was 76 ± 8 min in the test group with a DMFT of 6–10 and 258 ± 28 min in the control group.¹⁷ In the current investigation, a comparable time span was noted, with the test group lasting 40.50 ± 13.63 minutes and the control group lasting 279 ± 3.45 minutes.

The Oratest was found to be an efficient and cost-effective chairside tool for caries detection by Kunte SS et al. and Sundaram M et al. after they correlated it with the S. mutans laboratory culture test.

As the rate of caries increases, the absolute value of S. mutans also increases, which is evident by both, the laboratory culture as well as Saliva Check Mutans kit test. The increased bacterial count leads to lowering of the time taken by Oratest to test positive.

This study helps to identify and assess various chairside methods for detecting S. mutans count that can be meaningful educational and evaluation tools for high caries risk children. This can further be used as non-invasive methods for caries prevention by early detection and preventive program implementations. The Limitations of the study were (a) Sample size was less, (b) The GC Saliva Check Mutans kit is an expensive kit hence it limits its use for routine cases. This study requires a larger sample size in future to ascertain its use an educational and motivational tool for patients.

CONCLUSION

Within the limits of the study, it can be stated that all the tests correlated well with the DEFT scores of all subjects as well as with each other. The choice of the test is dependent upon its availability, cost-effectiveness, time constraints, and the requirements of the individual patient. The new GC Saliva Check Mutans kit is costly but is a wonderful tool for large scale S. mutans count evaluation as the time required and chances of error are very low as compared to the conventional laboratory method.

ORCID

Aakansha Sharma https://orcid.org/0000-0001-5192-8747

Nidhi Agarwal https://orcid.org/0000-0002-9231-8256

REFERENCES

1. Kunte SS, Chaudhary S, Singh A, et al. Evaluation and co-relation of the Oratest, colorimetric Snyder’s test and salivary Streptococcus mutans count in children of age group of 6–8 years. J Int Soc Prev Community Dent 2013;3(2);59–66. DOI: 10.4103/2231-0762.122433.

2. American Academy of Pediatric Dentistry. Definition of Early Childhood Caries. AAPD 2008;1.

3. Sundaram M, Nayak UA, Ramalingam K, et al. A comparative evaluation of Oratest with the microbiological method of assessing caries activity in children. J Pharm Bioall Sci 2013;5(Suppl 1):S5–S9. DOI: 10.4103/0975-7406.113283.

4. Gao XL, Seneviratne CJ, Lo EC, et al. Novel and conventional assays in determining abundance of Streptococcus mutans in saliva. Int J Paedtr Dent 2012;22(5):363–368. DOI: 10.1111/j.1365-263X.2011.01207.x.

5. Chaudhary SD, Chaudhary M, Singh A, et al. An assessment of the cariogenicity of commonly used infant milk formulae using microbiological and biochemical methods. Int J Dent 2011;2011:320798. DOI: 10.1155/2011/320798.

6. Bhasin S, Sudha P, Anegudi RT. Chair side simple caries activity test: Oratest. J Indian Soc Pedod Prev Dent 2006;24(2):76–79. DOI: 10.4103/0970-4388.26020.

7. Fontana M, Zero DT. Assessing patients’ caries risk. J Am Dent Assoc 2006;137(9):1231–1239. DOI: 10.14219/jada.archive.2006.0380.

8. Powell LV. Caries risk assessment: Relevance to the practitioner. J Am Dent Assoc 1998;129(3):349–353. DOI: 10.14219/jada.archive.1998.0209.

9. Eşian D, Man A, Burlibașa L, et al. Salivary level of Streptococcus mutans and Lactobacillus spp. related to a high a risk of caries disease. Rom Biotechnol Lett 2017;22(2):12496–12503.

10. Koneru S, Tanikonda R. Salivaomics – A promising future in early diagnosis of dental diseases. Dent Res J 2014;11(1):11–15. PMID: 24688554.

11. Lumikari ML, Loimaranta V. Saliva and dental caries. Adv Dent Res 2000;14:40–47. DOI: 10.1177/08959374000140010601.

12. Leal SC, Mickenautsch S. Salivary streptococcus mutans count and caries outcome – A systematic review. J Minim Interv Dent 2010;3(4):137–147.

13. Guo L, Shi W. Salivary biomarkers for caries risk assessment. J Calif Dent Assoc 2013;41(2):107–118. PMID: 23505756.

14. Twetman L, Twetman S. Comparison of two chair-side tests for enumeration of mutans streptococci in saliva. Oral Health Dent Manag 2014;13(3):580–583. PMID: 25284515.

15. Tal H, Rosenberg M. Estimation of dental plaque levels and gingival inflammation using a simple oral rinse technique. J Periodontol 1990;61(6):339–342. DOI: 10.1902/jop.1990.61.6.339.

16. Saxena S, Pundir S, Aena J. Oratest: A new concept to test caries activity. J Indian Soc Pedod Prev Dent 2013;31(1):25–28. DOI: 10.4103/0970-4388.112400.

17. Chandak S, Kesri R, Bhardwaj A. Milk, a simple tool to detect caries activity: Oratest. Int J Clin Prev Dent Res 2016;3(2):98–101. DOI: 10.5005/jp-journals-10052-0021.