Evaluation of Chair-Side Assays in High Microbiological Caries-Risk Subjects

Mutans and ClinproTM Cario L-PopTM in high microbiological caries-risk dental students compared with conventional semi-quantitative colony counting culture-based technique as the reference method. Saliva samples from 93 subjects of both sexes aged 18-26 years were seeded (Köhler and Bratthall method) on plates containing SB-20M culture medium method and 12 subjects with high caries risk were selected. These 12 individuals were subjected to determination of caries risk using two commercial rapid detection chair-side assays (Saliva-Check Mutans and ClinproTM Cario L-PopTM) according to the manufacturers’ instructions. The results were analyzed by the Kappa correlation test using SAS statistical software. There was a perfect agreement (Kappa=1) among the three caries risk evaluation methods chair-side assays and semi-quantitative CFU count (control) in all subjects. The results suggest that the commercial chair-side assays evaluated in this study may be practical and useful to identify high microbiological caries-risk subjects. Evaluation of Chair-Side Assays in High Microbiological Caries-Risk Subjects


Introduction
According to the Susan Fisher-Owens conceptual model, oral health is influenced by several layers of factors that act together and interact in a dynamic and complex way.Basically, this multilevel conception includes 5 major fields: genetics and biology; social environment; physical environment; health-influencing behaviors; and medical care; in individual, family and community levels, interacting across time and space (1).
Considering the biological determinants, microorganisms may cause an imbalance between tooth and plaque and play an important role in the development of dental caries.It is proposed that various species of microorganisms, such as mutans streptococci, lactobacilli, aciduric strains of non-mutans streptococci, Actinomyces, bifidobacteria and yeasts are involved in caries progression (2).
Mutans streptococci (MS) have been identified as the major pathogens of dental caries (2,3) because they are frequently present in caries lesions, are acidogenic and aciduric species (4), and are able to adhere to the tooth surface and other microorganisms (4).
Although the complexity of the contemporary concepts on dental caries and its manifestations hinders the establishment of a risk assessment model, an effective caries control and management requires the early identification of susceptible individuals for timely intervention.
Streptococcus mutans is an important biomarker of caries risk (5) because it has a key role in the development of dental caries.Detection of S. mutans in dental biofilm and saliva has been used as a manner to predict microbiological caries risk and monitor caries activity (6).The prevalence of S. mutans has been traditionally determined by microbial culture-based techniques (7)(8)(9)(10), but there are also commercially available chair-side assay kits for this purpose (5,11).
Saliva-Check Mutans (GC America, Alsip, IL, USA) immunoassay detection system was introduced to promote a rapid detection of S. mutans levels in saliva.According to the manufacturer, it uses a very specific immunochromatography process with two monoclonal antibodies that selectively detect only S. mutans.A positive result of the antigen-antibody reaction means that the subject has S. mutans levels equal to or above 500,000 colony forming units (CFU) per mL of saliva (12), which indicates risk for caries disease.With sensitivity and specificity above 90%, Saliva-Check Mutans has been considered as a promising tool for caries risk assessment in the community and clinical settings (5) and its efficacy either alone or combined with other chair-side assays has been demonstrated (5,11,13,14).Except from a recent study (11)  bacteria (15).The reaction is based on the enzymatic degradation of lactic acid by lactate dehydrogenase and coupled to a cascade of redox indicators that generate a color signal, which is analyzed using a semi-quantitative scale.Clinpro TM Cario L-Pop TM has been previously evaluated (16)(17)(18)(19) and, according to Schiffner and Torres-Quintero (16), it can indicate changes in the oral microflora and thus could be used to monitor the effect of interventions.
An advantage of using commercial kits for assessing microbiological caries risk is that these assays produce results shortly after the collection of saliva samples.In addition, the kits are easy to use in the daily clinical practice, without the need of laboratory procedures (11).Commercial chair-side assays can also be used to obtain information in community caries prevention/control studies.When traditional culture-based detection tests are used, results are normally obtained after 2 to 4 days because of the need of incubation and CFU counting procedures, which can be an inconvenient for patients, clinicians and public health workers (5).
The aim of this study was to evaluate the commercial chair-side assays Saliva-Check Mutans and Clinpro TM Cario L-Pop TM in high microbiological caries-risk subjects compared with conventional semi-quantitative colony counting culture-based technique as the reference method.

Material and Methods
Ninety-three 18-26-year-old undergraduate dental students of both sexes from the Dental School of the Universidad Nacional de Tucumán, San Miguel de Tucumán (Argentina) who did not use antimicrobial agents or antibiotics in the previous 3 months were invited to participate in the study.The study was approved by the Ethics Research Committee of the Medical School of the Universidad Nacional de Tucumán (Argentina).Approximately 2 h after toothbrushing and without any food ingestion, non-stimulated saliva samples were collected with a wooden spatula during 1 min, according to the Köhler and Bratthall (20) method and seeded on the surface of plates containing SB-20M culture medium selective for mutans streptococci (9,10).The plates were incubated for 3 days at 37 °C in microaerophilia using the candle jar technique for determination of the salivary levels of mutans streptococci.
The number of colony forming units (CFU) per milliliter of saliva was counted by a single calibrated examiner (Kappa>0.8)using a stereomicroscope (Nikon, Tokyo, Japan) with reflected light at 20x magnification.During examination, the plates were maintained against a dark background in order to highlight the characteristics of the colonies and facilitate identification.S. mutans colonies have a granular surface resembling ground glass, with or without a polysaccharide drop on the surface.Colonies with the typical morphology of S. mutans were randomly selected and transferred to tubes containing thioglycollate broth without glucose or indicator, which were incubated at 37 °C for 24 h for biochemical identification.The broth cultures were then used as inoculum to test the capacity of each isolate to ferment mannitol (with and without bacitracin), sorbitol, raffinose and melibiose (21) and to produce hydrogen peroxide (22).
After this initial screening (sample 1), the 12 students with salivary S. mutans levels greater than 100 CFU/mL, indicating high microbiological caries-risk according to Köhler and Bratthall (20) classification, were enrolled in the study and formed a convenience sample.After that, the participants received verbal explanation about the study purposes and procedures.The 12 individuals were subjected to caries risk assessment using the commercial chair-side assay kits Saliva-Check Mutans (GC America) and Clinpro TM Cario L-Pop TM (3M ESPE) and the results were compared with those obtained with the conventional semi-quantitative S. mutans colony counting culturebased technique (reference microbiological method).Both commercial kits were used according to the manufacturers' instructions.
For microbiological caries risk assessment with Saliva-Check Mutans assay, a paraffin-stimulated whole-saliva sample was collected from each individual (sample 2) and one drop of #1 reagent was added.The container was tap 15 times and then four drops #2 reagent were added.Saliva was shaken until it turned green.Three drops of the saliva with reagents were dispensed in the sample window of the test device, with a 15-minute waiting time.Positive result was obtained if either a faint or clear red line appeared, indicating the presence of over 500,000 cfu/mL of S. mutans.
For microbiological caries risk assessment with Clinpro TM Cario L-Pop TM test, the subjects were first asked to brush their teeth for 1 min with the toothbrush and dentifrice used routinely.Five minutes later, a cotton-tipped applicator provided by the manufacturer was rubbed four times on the dorsum of the tongue using rotating movements to soak the applicator with saliva (sample 3).Next, the applicator was introduced into a device containing specific reagents that produce enzymatic degradation of the lactic acid present in saliva, and left in contact 2 min.After this time, the color change in the applicator tip was compared and matched with a specific color scale that comes in the kit.The results of this assay were validated by a control test made with a black applicator supplied by the manufacturer.Each color grade in the scale is related to scores from 1 to 9. Scores 1 to 3 indicate low lactic acid production; scores 4 to 6 indicate a moderate production, and scores 7 to 9 indicate high lactic acid production, and indicate low, medium and high caries risk, respectively.
The results obtained in the assays were analyzed by the Kappa correlation test using the SAS (Statistical Analysis System) statistical software for Windows v. 9.1.3(SAS Institute Inc., Cary, NC, USA).

Results
Table 1 presents the mean results of the analysis of three saliva samples obtained from of each of the 12 subjects with high caries risk, determined by counting more than 100 CFU/mL of saliva in the semi-quantitative colony counting culture-based technique.Positive reaction was observed in all subjects (100%) after application of the Saliva-Check Mutans assay, confirming the presence of anti-mutans antibodies and the presence of over 500,000 S. mutans CFUs per mL of saliva.Scores between 7 and 9 were obtained with the Clinpro TM Cario L-Pop TM assay.Score 7 was observed in 8.3% of the cases, score 8 in 50% and score 9 in 41.7%, which means that all subjects presented high levels of lactic acid production and should therefore be classified as having high microbiological risk for dental caries.
There was a perfect agreement (Kappa=1) among the three methods of microbiological caries risk assessment -chair-side assays and conventional semi-quantitative colony counting culture-based technique (reference method) -in all subjects with high caries risk (100% of the cases).

Discussion
Due to the polarized distribution of dental caries among a high-caries risk minority, 20-25% of children account for over 50% of all caries lesions (23).In this way, targeting prevention and interventional measures to high caries risk populations is a rational approach for achieving a cost-effective dental caries control (24).Although it is known that caries disease involves a complex, multilevel and multifactorial concept, the main focus of this study was high microbiological caries risk.For this reason, this study was performed only with individuals considered to be at high microbiological risk for caries.
In this study, the participants were selected using the wooden spatula method proposed by Köhler and Bratthall (20) (conventional microbial culture technique), according to which individuals with more than 100 S. mutans CFUs per saliva mL are considered at high microbiological risk for dental caries.
The objective of this study was to evaluate if the results of two widely used commercial chair-side assays -Saliva-Check Mutans and Clinpro TM Cario L-Pop TM -agree with those of the conventional detection of microbiological caries risk (semi-quantitative colony counting culturebased technique) in highly infected individuals by S. mutans.
Using commercial kits allows detecting the microbiological caries risk in the daily clinical practice in a practical and fast way, without the need of laboratory procedures (11).
The introduction of the Saliva-Check Mutans assay allowed for bringing a molecular detection method into practical applications at chair-side since the dentist can perform all the procedures.Culture, additional apparatus or special facilities are not required and there are no expenses for laboratory costs.Because of its high correlation with polymerase chain reaction-confirmed CFU/mL of S. mutans and its compatibility with other commercial products, Saliva-Check Mutans appears to be suitable for use in a dental clinic, easy to use and rapid in providing results.It could be used, for example, for screening children with high S. mutans levels at primary schools (13).According to Gao et al. (5), the monoclonal antibody-based immunoassay accurately and rapidly determines abundant S. mutans salivary levels, and is appropriate for microbiological chairside caries risk assessment.
Wennerholm and Emilson (11) evaluated the combination of two rapid semi-quantitative detection kits (Saliva-Check Mutans and Saliva-Check IgA Mutans) to assess caries risk compared with the Cariogram computer-based program.(20).
In view of the good agreement with the Cariogram, those authors concluded that these commercial kits could be used for microbiological caries risk assessment.The results agree with those of the present study, in which Saliva-Check Mutans test was able to adequately classify microbiological high-caries-risk patients, using conventional microbial culture technique as the reference method.
Clinpro TM Cario L-Pop TM evaluates microbiological caries risk in an indirect manner, since it does not enumerate CFUs, but rather detects the presence of a bacterial product -lactic acid.The darkness of the color signal reflects the ability of dental plaque to produce acids and thus promote the caries process.The higher the metabolic activity of the acidogenic bacteria, the higher their potential to promote caries development (15).It is not, therefore, a specific test for S. mutans, since it detects lactic acid production by all bacteria from the oral microbiota that can produce lactate.However, the results with the use of Clinpro TM Cario L-Pop TM in this study showed an association with high salivary levels of S. mutans.In a clinical trial, Gerardu et al. (2006) (17) concluded that Clinpro TM Cario L-Pop TM can be used to monitor and motivate compliance to oral hygiene.In the present study, the results obtained with Clinpro TM Cario L-Pop TM confirmed those obtained with the conventional culture-based method in all cases, showing a perfect agreement.Further studies are required, including longitudinal investigations and studies that have a larger sample size and assess the influence of other aspects of caries risk than microbiological ones.
Within the limitations of this study it may be concluded that Saliva-Check Mutans and Clinpro TM Cario L-Pop TM assays showed perfect agreement in comparison with the conventional semi-quantitative colony counting culturebased technique as the reference method.The results indicate that both commercial chair-side assays may be practical and useful to identify high microbiological caries risk subjects.