Efficacy of a dip slide test for mutans streptococci in caries risk assessment

Abstract – The aim of this study was to evaluate the efficacy of a dip slide test for mutans streptococci in caries risk assessment, when the microbiological results were compared to well‐defined clinical criteria (DCC) for caries risk, clinically measured through high and low caries activity. Eighty‐one volunteers from the 6^th to 8^th grades from public schools of Piracicaba, São Paulo, Brazil, were evaluated for dental caries. All free smooth surfaces were evaluated to check whether or not there were white spots. Based on the subjects’ caries experience, a calibrated clinician divided them into groups of high and low caries activity. The subjects were submitted to a salivary test (CARITEST SM^®) from the same batch number. Kappa statistics (κ) were applied to verify the reproducibility of the simplified test, checked through interexaminer agreement when the results were classified by independent and blind means. The microbiological results were validated according to expressions of sensitivity and specificity. A moderate agreement was verified as the results were classified according to 6 scores (κ=0.55), and the agreement was substantial when the results were classified according to high and low microbiological count (κ=0.78). The sensitivity and specificity values were 0.59 and 0.85, respectively, showing that the test was more specific than sensitive, and could thus better identify the low caries risk subjects.     

Detection of mutans streptococci by latex agglutination test and its application as a caries-activity test

The number of mutans streptococci in saliva and dental plaque has been reported to correlate with the incidence of dental caries. This report describes a simple and rapid diagnostic method for the detection of mutans streptococci in dental plaque using latex agglutination (LA) test. Latex particles (0.876 microns, diameter) were sensitized with partially purified antibodies against whole cells of Streptococcus mutans MT8148 (c), MT703R (e) and OMZ175 (f) and Streptococcus sobrinus B13 (d) and 6715 (g). Whole cells of mutans streptococci or dental plaque was extracted with a mixture of 8M sodium nitrite (5 microliters) and 2M acetic acid (5 microliters) for five minutes and neutralized with 2M sodium hydroxide (10 microliters), and the extract and the sensitized latex suspension (20 microliters) were mixed and the grade of agglutination reaction was read macroscopically after ten minutes standing at room temperature. The LA tests could detect 1.0 10ng of purified serotype antigen and 10(5)-10(6) CFU of live cells of mutans streptococci and specifically distinguish not only the mutans streptococci from the other streptococci but also S. mutans from S. sobrinus. However, cross-reactions were still observed among the serotypes c, e and f of S. mutans or between the serotypes d and g of S. sobrinus. Plaque samples were collected from 168 children (2 to 12 years of age) and the 0.1 mg (wet weight) were applied to the LA tests. At the same time, the total number of mutans streptococci in plaque and the serotypes of each isolate were determined. The results of LA reaction correlated significantly with the number of mutans streptococci in plaque (chi-square analysis; p less than 0.0001). The LA tests discriminated between S. mutans and S. sobrinus in dental plaque. It was found that the latex particles sensitized with anti-serotype c and/or e S. mutans antibodies were most effective in demonstrating mutans streptococci, and they were used in the following studies. The results of LA reaction correlated significantly with both the number of decayed tooth surfaces and the number of decayed and filled tooth surfaces. These results suggest that the LA test in mutans streptococci can be useful for the detection of mutans streptococci in dental plaque and also as a caries-activity test at dental clinics.     

Transmission of mutans streptococci.

It has been shown that early establishment of mutans streptococci in the mouth of infants increases risk of caries. Extensive studies on the timing of infection, and on sources and routes of transmission of the organism have been performed. Results of many studies suggest that the source of mutans streptococci is the mother, especially her saliva. Methods used to attempt to verify this hypothesis have developed considerably in recent years. Recent research and work in progress using methods based on DNA analysis have increased and will extend knowledge about transmission. This review summarizes the results obtained by different methods of transmission of mutans streptococci. The final goal of studies is to accumulate adequate information for prevention strategies of mutans streptococcal infection.     

Prevalence of mutans streptococci in one-year-old children

Colonization with mutans streptococci was studied in 1095 1-year-old children living in suburban Stockholm. During a scheduled vaccination appointment at a child health centre, a bacterial sample was obtained from the child's tongue and a structured questionnaire was completed by the accompanying parent. Six percent of the subjects were colonized with mutans streptococci. The variables most strongly correlated with presence of mutans streptococci were: non-Swedish background, consumption of sugar-containing beverages at night and total consumption of sugar-containing beverages. The results indicate that, by the age of 1 year, maternally influenced behaviour patterns such as dietary habits that may predispose to early colonization of mutans streptococci are already established. Such early colonization with mutans streptococci may predict high caries risk in the primary dentition.     

Acquisition and transmission of mutans streptococci

Dental caries is an infectious and transmissible disease. The mutans streptococci and some Lactobacillus species are the two groups of infectious agents most strongly associated with dental caries. Earlier studies demonstrated that infants acquire mutans streptococci from their mothers and only after the eruption of primary teeth. More recent studies indicate that mutans streptococci can colonize the mouths of predentate infants and that horizontal, as well as vertical, transmission does occur. These findings will likely facilitate the development of clinical strategies to prevent or delay infant infection by these organisms, thereby reducing the prevalence of dental caries.     

Latex agglutination test for detection of mutans streptococci in relation to dental caries in children.

A simple and rapid system based on a latex agglutination (LA) reaction was devised for the detection of mutans streptococci in dental plaque. Latex particles were sensitized with antibodies against whole cells of Streptococcus mutans strains MT8148 (serotype c), MT703R (e) and OMZ175 (f) and Strep. sobrinus strains B13 (d) and 6715 (g). These sensitized particles were agglutinated within a few minutes after addition of 1.0-10 ng serotype-specific antigen from the homologous organisms or the nitrous acid extract of whole cells at 10(5)-10(6) c.f.u. The LA test specifically differentiated not only mutans streptococci from the other oral streptococci but also Strep. sobrinus from Strep. mutans. The LA test was also applicable to extracts of plaque from 206 human subjects who harboured mutans streptococci. In clinical trials, the outcome of the LA test correlated significantly with the number of mutans streptococci found in plaque (p less than 0.0001), which was quantified by the selective cultivation of mutans streptococci. Furthermore, the LA test discriminated between Strep. mutans and Strep. sobrinus from human dental plaque. The sensitivity and the specificity of the LA test for detection of mutans streptococci were 78.9 and 100%. The degree of reactivity in the LA test correlated significantly with the number of decayed tooth surfaces (p less than 0.0001) and decayed and filled tooth surfaces (p less than 0.0001). These results suggest that the LA test could be useful clinically for the detection of mutans streptococci in dental plaque as well as serving as a caries-activity test.     

Antibody responses to mutans streptococci in children.

Because mutans streptococci (Streptococcus mutans, S. sobrinus) are considered the main causative bacteria in human dental caries, immune responses to these bacteria have aroused much research interest over the last two decades. Studies in man have focused mainly on salivary and serum antibodies developing naturally in response to oral colonization by mutans streptococci, or in relation to the development of dental caries. Although both salivary (IgA) and serum-derived (IgG) antibodies have been shown in many studies to protect against the adherence of and to interfere with the metabolism of mutans streptococci, no conclusive evidence relating to their clinical significance is available. In young children, serum IgG antibodies to S. mutans seem more important than salivary IgA antibodies in relation to protection against dental caries. In studies in animals and, recently, in man, monoclonal IgG antibodies to S. mutans protein antigen I/II ("adhesin") have provided effective protection against mutans streptococci. Whether they could also prevent dental caries in man is not yet known.     

Cariogenic traits in xylitol-resistant and xylitol-sensitive mutans streptococci

Long-term xylitol consumption leads to the emergence of xylitol-resistant (X-R) mutans streptococci. The aim of the present study was to compare cariogenic traits in X-R and xylitol-sensitive (X-S) strains. Six strains of mutans streptococci, three X-R and three X-S strains, were studied. Xylitol resistance and sensitivity were confirmed by growth in xylitol-supplemented media. Acid production from glucose or fructose or uptake of xylitol was initiated by adding (14)C-labelled glucose, fructose or xylitol to bacterial suspensions. The resultant metabolites were identified by HPLC. Lactate was the major metabolite from glucose, whether the bacteria were grown in the presence or the absence of xylitol. Lactate production per colony-forming unit was lower in X-S cells than in X-R cells. Fructose was metabolized by both X-R and X-S cells. Both X-R and X-S cells took up xylitol, but xylitol-5-P was detected in X-S cells only. Total polysaccharides were measured through production of C(14)-labelled ethanol-insoluble polymers from [U(14)-C]-sucrose. No difference in polysaccharide production was found between X-R and X-S cells. The present study thus does not support the contention that X-R are less cariogenic than X-S mutans streptococci.     

Longitudinal study of intrafamilial mutans streptococci ribotypes

To explore the intrafamilial homology and longitudinal stability of colonization by early acquired mutans streptococci, genomic fingerprinting was performed on 254 strains (192 Streptococcus mutans and 62 Streptococcus sobrinus strains) collected from 16 families (16 mother-child pairs, seven fathers and four siblings). Genomic DNA was digested by the restriction endonuclease HindIII, followed by gel electrophoresis, Southern blotting, and hybridization with a digoxigenin-labeled 16S rDNA probe, and hybrid detection by enhanced chemiluminescence. One to five ribotypes were identified per person, and between two and nine (median five) within each family. Fourteen of the 16 mother-child pairs showed homology for at least one ribotype (range 1-4). Six of the seven father-child pairs had one ribotype in common. Ten of the 13 longitudinally examined children showed persistence of at least one ribotype over a period of up to 16 yr. The results support the notion of intrafamilial transfer of mutans streptococci, and suggest that colonization of early acquired strains persists into young adulthood.     

Intraindividual variations in counts of mutans streptococci measured by "Strip mutans" method

Abstract— Intraindividual variations in the mutans streptococci measured by Strip mutans, in a 1-day and a 5-day period, as well as the effect of toothbrushing, were investigated. 30 subjects participated, and their levels of salivary mutans were estimated by a scale containing four steps. With the exception of three comparisons, time intervals of different lengths did not cause a deviation of more than one step. Comparisons made before and after toothbrushing yielded the same score in 22 cases, whereas two subjects showed a discrepancy of two steps. Clearly, in a short-term perspective, variations can certainly be found, but more pronounced discrepancies are rare.     

Intraindividual variations in counts of mutans streptococci measured by "Strip mutans" method

Intraindividual variations in the mutans streptococci measured by Strip mutans, in a 1-day and a 5-day period, as well as the effect of toothbrushing, were investigated. 30 subjects participated, and their levels of salivary mutans were estimated by a scale containing four steps. With the exception of three comparisons, time intervals of different lengths did not cause a deviation of more than one step. Comparisons made before and after toothbrushing yielded the same score in 22 cases, whereas two subjects showed a discrepancy of two steps. Clearly, in a short-term perspective, variations can certainly be found, but more pronounced discrepancies are rare.     

奶瓶龋儿童口腔变链菌的研究

目的：研究儿童口腔内的变链菌基因型与奶瓶龋易感性的关系。方法：取20名奶瓶龋及20名无龋儿童牙菌斑及唾液接种于TYCSB培养基计数并筛选MS,采用AP－PCR法鉴定变链菌基因型。结果：奶瓶龋组儿童唾液及菌斑变链菌计数高于无龋组;并且有两种以上基因型变链菌的比例也高于对照组（14／20vs 5/20)。结论：患有奶瓶龋的儿童不仅变链菌计数高,而且多数被两个以上基因型的MS感染。     

Maintaining mutans streptococci suppression with xylitol chewing gum

BACKGROUND: One strategy for treating dental caries is to suppress oral mutans streptococci, or MS, with chlorhexidine, or CHX, mouthrinse. Oral MS levels, however, tend to quickly return to baseline values without further intervention. In this clinical study, the authors evaluated the effect of xylitol chewing gum on MS regrowth. METHODS: The authors selected 151 subjects with elevated oral MS levels (> or = 105 colony-forming units per milliliter, or CFU/mL, of paraffin-stimulated saliva). Subjects rinsed with 0.12 percent CHX gluconate mouthrinse twice daily for 14 days. The authors then randomly assigned the subjects to one of three groups. Those in the test group (n = 51) chewed a commercial xylitol gum three times daily for a minimum of five minutes each time for three months. The placebo group subjects (n = 50) used a commercial sorbitol gum, and the control group subjects (n = 50) did not chew gum. The authors estimated MS load on the dentition using paraffin-stimulated saliva samples. The authors serially diluted the samples, plated them on selective media and incubated them anaerobically; they then enumerated the colonies under a stereomicroscope. RESULTS: MS levels were not significantly different between the three groups at baseline (mean log CFU/mL +/- standard deviation: 5.4 +/- 0.7, 5.4 +/- 0.8, 5.2 +/- 0.7, respectively) nor after CHX therapy (2.7 +/- 0.8, 3.1 +/- 1.1, 3.0 +/- 1.1, respectively). After three months of gum chewing, the test group subjects had significantly lower salivary MS levels (3.6 +/- 1.2) than did the placebo (4.7 +/- 1.2) or control (4.4 +/- 1.3) group subjects. CONCLUSIONS: Xylitol chewing gum appears to have the ability to prolong the effect of CHX therapy on oral MS. CLINICAL IMPLICATIONS: Maintaining long-term caries-pathogen suppression is feasible with currently available commercial products and can be expected to result in significant caries inhibition.     

Genetic diversity of plaque mutans streptococci with rep-PCR

Mutans streptococci (MS) are key organisms associated with the etiology of dental caries. Using probabilities that were tested by oversampling, we designed this study to determine the minimal number of MS isolates from an individual required to evaluate diversity of genotypes. MS isolates were genotyped by repetitive extragenic palindromic-polymerase chain-reaction (rep-PCR). Analysis of 20 isolates from individuals resulted in a mean of 1.6 and 2.4 genotypes in children (N = 12) and adults (N = 10), respectively. In a follow-up study, reducing the number of isolates to 7-10 resulted in a theoretical probability of up to 78% for detecting up to 4 genotypes. A mean of 1.5 genotypes was found in 35 children and 10 adults. These findings provide evidence for the design of studies of MS genotyping that can serve as a model for the analysis of genotypes within individuals.     

Distribution of mutans streptococci in families: a longitudinal study

The purpose of this study was 1) to investigate whether a group of children not colonized by mutans streptococci (MS) at the age of 3 years were colonized several years later, and 2) to study whether MS that appeared in the children were identical to those found in the parents. In a previous study no MS were found in 13 3-year-old first-born children. In 10 of these children pooled plaque samples were again collected after 5 years, and in the other 3 children after 2 years. Additionally, separate plaque samples were obtained from the children's first permanent molars when the teeth were present. Pooled samples were also obtained from all the parental pairs at follow-up and from three pairs at baseline. MS were isolated, and genotyping was done through DNA fingerprinting by restriction endonuclease analysis. In 10 children MS were still not found. Among the three children with detectable MS the DNA fingerprints of the bacteria were the same in one girl and her mother and in one boy and his father; in the other boy no similar pattern was found in either mother or father. None of the individuals in the 13 parental pairs showed identical genotypes of MS. The results indicate an opportunity to remain MS-negative if not colonized at the age of 3 years. The difficulty of being colonized with MS from the spouse is highlighted.     

Long-term xylitol consumption and mutans streptococci in plaque and saliva

The effect of 4 to 6 years of habitual use of xylitol gum (greater than or equal to 2 times per day) on the viable counts of mutans streptococci in both plaque and saliva was evaluated in a cross-sectional study of participants of the Ylivieska xylitol study (Finland, 1982-1984). Paraffin-stimulated saliva was collected first, whereafter the remaining plaque was collected from all available tooth surfaces and pooled. The habitual xylitol consumers (n = 15) had less plaque than the controls (n = 21). The total viable counts of mutans streptococci in the plaque collected from the habitual xylitol users were about 10% of those determined in controls (p less than 0.01). The salivary mutans streptococci levels were relatively high in both habitual xylitol consumers and controls, as determined using a plate count and with an adhesion-based assay; no significant differences were detected between these groups. Thus, habitual xylitol consumption appeared not to be associated with a reduction in the total numbers of mutans streptococci in saliva. The plaque and the mutans streptococci were, however, more loosely bound to the tooth surfaces of the habitual xylitol users. The results of the plate count and the adhesion-based assay for salivary mutans streptococci were in good agreement for both habitual xylitol consumers and the controls. This suggests that it was the accumulation of plaque rather than the adhesiveness of mutans streptococci that was affected by the habitual consumption of xylitol.