The effectiveness of enzymic irrigation in removing a nutrient-stressed endodontic multispecies biofilm.

AIM: To establish a nutrient-stressed multispecies model biofilm and investigate the dynamics of biofilm killing and disruption by 1% trypsin and 1% proteinase K with or without ultrasonic activation. METHODOLOGY: Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown on hydroxyapatite discs and in prepared root canals of single-rooted teeth in modified fluid universal medium. The treatment groups included trypsin, proteinase K, 0.2% chlorhexidine gluconate and 1% sodium hypochlorite (NaOCl) (with and without ultrasonics). NaOCl and chlorhexidine were the positive controls and untreated group, and sterile saline was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture. RESULTS: Nutrient stress in the multispecies biofilm was apparent as the medium pH became alkaline, glucose was absent, and serum proteins were degraded in the supernatant. The CLSM showed the percentage reduction in viable bacteria at the biofilm surface level due to nutrient starvation. On the disc model, trypsin and proteinase K were effective in killing bacteria; their aerobic viable counts were significantly lower (P < 0.01) than the negative control and chlorhexidine. NaOCl was the most effective agent (P < 0.001). In the tooth model, when compared to saline, trypsin with ultrasonics caused significant killing both aerobically and anaerobically (P < 0.05). Chlorhexidine (1.46 ± 0.42), trypsin (3.56 ± 1.18) and proteinase K (4.2 ± 1.01) with ultrasonics were significantly effective (P < 0.05) in reducing the substratum coverage as compared to saline with ultrasonics (12% ± 4.9). CONCLUSION: Trypsin with ultrasonic activation has a biofilm killing and disrupting potential.

Dental caries from a molecular microbiological perspective.

Dental caries results from an imbalance of the metabolic activity in the dental biofilm. The microbial communities of teeth have traditionally been studied by standard cultural approaches. More recently, cloning and sequencing of the 16S rRNA gene have been used to characterize the microbial composition of the oral biofilm, but the methodological limitations of this approach have now been recognized. Next-generation high-throughput sequencing methods have the potential to reveal the composition and functioning of the biofilm by means of metagenomic and metatranscriptomic analyses. Currently available high-throughput sequencing approaches are reviewed and discussed in relation to studying the biofilm associated with dental caries. Important in understanding the dynamic processes in caries is the metabolic activity of the biofilm; metabolome analysis is a new tool that might enable us to assess such activity. As caries is a localized disease, it is essential that biofilm samples are taken from precisely determined tooth sites; pooling samples is not appropriate. This paper presents the case that culture-based studies are important, but that the fullest understanding of the role of the biofilm in the caries process will only come from an integrated approach determining biological function and metabolic output.

Multidisciplinary research agenda for novel antimicrobial agents for caries prevention and treatment.

Antimicrobial methods to augment fluoride-mediated caries inhibition are necessary. Several methods are described here, but none was considered likely to be as effective as fluoride usage. None had been tested in effective models to demonstrate their ability to act either additively or synergistically with fluoride-containing toothpastes. Dental caries is a biofilm-mediated disease: The composition of the biofilm associated with caries initiation and progression is diverse. Caries-associated taxa - including mutans streptococci, lactobacilli, bifidobacteria, and yeasts - may be useful surrogate markers for in vivo investigations. In vitro testing should progress from single-species planktonic cells to multi-species biofilms prior to essential testing in randomized control trials (RCTs). Modern high-throughput sequencing techniques need to be applied to the study of bacterial acquisition from birth and of the composition of the biofilm associated with the formation of white-spot lesions. The determination of the functions of the biofilm and the phenotype of the bacterial components may be determined by RNA-seq techniques, since they must be conserved between caries lesions and will include the ability to produce acids and survive and proliferate in acidic conditions. The application of such methods will significantly improve our understanding of the etiology and progression of dental caries.

The potential benefits of sugar-free chewing gum on the oral health and quality of life of older people living in the community: a randomized controlled trial.

OBJECTIVES: To determine the effects of prescribing sugar-free chewing gum on the oral health and quality of life of dentate older people living in the community and attending for routine dental care. METHODS: A randomized controlled trial was conducted on 186 older people who were not regular chewers of gum, (aged 60 years and over with ≥ 6 teeth) recruited from primary care clinics. Participants were randomly allocated to a gum-chewing group (chewing xylitol-containing gum twice a day for 15 min; n = 95) or a control group (no gum; n = 91). Both groups were examined at baseline and at the end of the study (6 months later). The primary outcome measure for the study was increased in stimulated saliva flow rate. Secondary measures included improvements in Plaque and Gingival Indices, and self-perceived change in oral health. RESULTS: The retention rate for the study was 78.5% (n = 146 at follow-up); reported compliance with the protocol was 84% (ranged between 12% and 100%). There was no significant change in the saliva flow of the gum-chewing group (1.20-1.17 ml/min), while the control group experienced an increase in flow rate (1.06-1.32 ml/min; P = 0.001). The gum-chewing group, however, demonstrated significant improvement in Plaque and Gingival Index scores over the control group. For the Plaque Index, the mean scores (±SD) were 0.29 (±0.29) and 0.56 (±0.46) for the gum-chewing group and control groups, respectively (P < 0.001), at the second examination, which remained significant after controlling for age and saliva flow rate. For the Gingival Index, the scores were 0.73 (±0.30) and 0.92 (±0.32), respectively (P < 0.001), which persisted after controlling for age. A significantly higher proportion of participants in the gum-chewing group perceived that their oral health had improved during the study period in comparison with the control group (40% cf 21%; P = 0.016). CONCLUSIONS: Prescription of sugar-free chewing gum to dentate older people living in the community and attending routine dental services was not associated with a significant increase in stimulated saliva flow. There were, however, significant improvements in Plaque and Gingival Index scores, and in self-perceived oral health.

Fluoride-supplemented milk inhibits acid tolerance in root caries biofilms.

In this study we investigated the effect of fluoride on plaque acid tolerance. The test group consumed 200 ml of milk supplemented with 5 mg F/l as NaF once a day, the milk control group drank 200 ml of unsupplemented milk, and the no-milk control group did not consume milk in this manner. Plaque samples were taken at baseline and after 15 months. The proportion of acid-tolerant bacteria in plaque was estimated using LIVE/DEAD® BacLight™ staining after exposure to pH 3.5 for 2 h. The fluoride group showed a statistically significant decrease in plaque acid tolerance compared to baseline. This study shows that daily intake of fluoride in milk reduces plaque acid tolerance.

Clonal structure of Streptococcus sanguinis strains isolated from endocarditis cases and the oral cavity.

A collection of Streptococcus sanguinis strains from patients with endocarditis (n = 21) and from the oral cavity (n = 34) was subjected to a multi-locus sequence typing analysis using seven housekeeping genes, carbamoyl-phosphate synthetase (carB), Co/Zn/Cd efflux system component (czcD), d-alanyl-d-alanine ligase (ddl), DNA polymerase III (dnaX), glucose-6-phosphate dehydrogenase (gdh), DNA-directed RNA polymerase, beta subunit (rpoB) and superoxide dismutase (sodA). The scheme was expanded by the inclusion of two the putative virulence genes, bacitracin-resistance protein (bacA) and saliva-binding protein (ssaB), to increase strain discrimination. Extensive intra-species recombination was apparent in all genes but inter-species recombination was also apparent with strains apparently harbouring gdh and ddl from unidentified sources and one isolate harboured a sodA allele apparently derived from Streptococcus oralis. The recombination/mutation ratio for the concatenated housekeeping gene sequences was 1.67 (95% confidence limits 1.25-2.72) and for the two virulence genes the r/m ratio was 3.99 (95% confidence limits 1.61-8.72); recombination was the major driver for genetic variation. All isolates were distinct and the endocarditis strains did not form distinct sub-clusters when the data were analysed using ClonalFrame. These data support the widely held opinion that infecting S. sanguinis strains are opportunistic human pathogens.

Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms.

AIM: To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm. METHODS AND RESULTS: Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a period of exposure to 20% sucrose solution (28 days), using Rogosa Agar. The lactobacillus colonies were randomly selected (n = 222) and subcultured. The isolates were identified using pheS or rpoA gene sequence analysis. Lactobacilli identified as Lact. paracasei (n = 75) were subjected to multilocus sequencing typing (MLST) analysis by determining partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG. An increase recovery of lactobacilli after sucrose phase compared with nonsucrose period was observed (31 prior to and 191 following a sucrose exposure period). Seven subjects harboured Lact. paracasei and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. CONCLUSION: The present data supports the hypothesis that oral lactobacilli may be of exogenous origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The study allow us to gain insight into the genetic diversity of Lact. paracasei in oral biofilm.

Resistance to acidic environments of caries-associated bacteria: Bifidobacterium dentium and Bifidobacterium longum.

Oral Bifidobacteriaceae, Bifidobacterium dentium and Bifidobacterium longum, are known to be isolated together with mutans streptococci and lactobacilli from caries lesions, suggesting that these Bifidobacteriaceae are caries associated and acid resistant. This study aimed to investigate effects of acidification on B. dentium and B. longum, and to compare them with those on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus paracasei. Effects of acidification, growth ability in a complex medium at a pH of 4.0-8.0, cell viability in 2-morpholinoethanesulfonic acid monohydrate (MES)-KOH buffer at pH 4.0, as well as stability of intracellular pH (pH(in)) at an extracellular pH of 3.5-8.0 estimated using a fluorescent dye, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester in MES-KOH, 3-(N-morpholino)propanesulfonic acid-KOH or N,N-bis(2-hydroxyethyl)glycine-KOH buffer, were investigated. B. longum grew as well as Streptococcus strains over a wide pH range, whereas B. dentium grew best in the narrow pH range around neutral. The cell viability of B. dentium decreased significantly after 2 h of acidification at a pH of 4.0, but this was significantly less than that of the Streptococcus and Lactobacillus species, whereas B. longum maintained almost 100% viability. The pH(in) was close to the extracellular pH at pH of 5.5-7.5 in the Bifidobacterium and Streptococcus strains, while at a pH of <5.0, the pH(in) was higher than the extracellular pH in all the strains, but the pH(in) maintenance ability of Bifidobacterium strains was higher than that of the Streptococcus strains. The high survival rate and pH(in) maintenance ability of bifidobacteria comparable to that of S. mutans in the acidic environment may account for why bifidobacteria exist as stable species in acidic caries lesions together with mutans streptococci.

Non-oral bifidobacteria and the aciduric microbiota of the denture plaque biofilm.

The microbiota of the denture plaque biofilm colonizing the fitting surface of dentures in edentulous subjects with healthy palates (n = 20) and in edentulous subjects with denture stomatitis (n = 20) was studied. The numbers of bacteria colonizing the dentures of healthy subjects was significantly less than the numbers colonizing the dentures of stomatitis subjects. The proportions and frequency of isolation of mutans streptococci, lactobacilli, bifidobacteria and yeasts were significantly (P < 0.05) greater in the subjects with denture stomatitis. The proportions of these organisms in the denture plaque biofilm of the subjects with denture stomatitis were similar to those found in carious lesions, indicating that the site is a low pH environment. The predominant bifidobacterial species in the mouths of dentate subjects is Bifidobacterium dentium but in the edentulous subjects wearing dentures B. dentium was isolated from only one of the 20 subjects with denture stomatitis and from none of the 20 subjects with healthy palates. Instead, Bifidobacterium breve, Bifidobacterium scardovii and Bifidobacterium longum subsp. longum were isolated. Only a single non-oral bifidobacterial species was isolated from each individual and repetitive extragenic palindromic- and BOX-polymerase chain reaction typing methods indicated that the same genotypes were shared between subjects. Using deferred antagonism spot plate assays, interspecies inhibition was demonstrated between oral isolates of B. dentium, B. breve, B. scardovii and B. longum subsp. longum. Here we have shown that bifidobacteria and caries-associated microbiota are present in denture plaque at levels similar to those of carious lesions and B. dentium cannot be maintained in an edentulous mouth.

Oral Bifidobacteria: caries-associated bacteria in older adults.

Bifidobacteria are aciduric bacteria that might play a role in the caries process. To test the hypothesis that Bifidobacteria behave as caries-associated organisms, as predicted by the ecological plaque hypothesis, we determined salivary levels of Bifidobacteria and caries-associated organisms for 156 older adults. Salivary levels of Bifidobacteria, mutans streptococci, lactobacilli, and yeasts were correlated with each other (p < 0.001), negatively correlated with salivary flow rate (p < 0.001), and positively correlated with plaque index (p < 0.05). Salivary Bifidobacteria levels were positively associated with the number of filled (p < 0.001) and decayed (p = 0.036) tooth surfaces and negatively associated with number of teeth (p < 0.001) and salivary flow rate (p = 0.049). In regression analyses, caries experience was significantly associated with only salivary Bifidobacteria (p < 0.001) and yeast (p < 0.001) levels and the individual's age (p = 0.021). Bifidobacteria should be regarded as caries-associated organisms whose role in the caries process and as markers of caries risk requires further investigation.

The effectiveness of passive ultrasonic irrigation on intraradicular Enterococcus faecalis biofilms in extracted single-rooted human teeth.

AIM: To compare the efficacy of passive ultrasonic irrigation with 1% sodium hypochlorite, with that of conventional syringe irrigation with 1% sodium hypochlorite, on intraradicular Enterococcus faecalis biofilms in extracted single-rooted human teeth. METHODOLOGY: Biofilms of E. faecalis (strain OMGS 3202) were grown on the prepared root canal walls of 48 standardized root halves which had been longitudinally sectioned. Following reapproximation, the roots were divided into four groups of twelve. The two experimental groups were treated with conventional syringe irrigation with 1% sodium hypochlorite solution (experimental group A) and passive ultrasonic irrigation with 1% sodium hypochlorite solution (experimental group B). Of the two control groups, the first was treated with conventional syringe irrigation with sterile saline solution (control group C), whilst the second control group (D) received no irrigation. The root halves were processed for scanning electron microscopy. Three images (x 700), coronal, middle and apical, were taken of the twelve root halves in each of the four groups, using a standardized protocol. The images were randomized and biofilm coverage assessed independently by three calibrated examiners, using a four-point scoring system. RESULTS: There were no significant differences in the scores for remaining biofilm coverage between group A (conventional syringe irrigation with 1% sodium hypochlorite) and group B (passive ultrasonic irrigation with 1% sodium hypochlorite) at the three observed levels. There was a significant difference between both experimental groups (groups A and B) and group C (conventional syringe irrigation with sterile saline solution) (P < 0.001) at all three observed levels. CONCLUSIONS: Both conventional syringe irrigation and passive ultrasonic irrigation with 1% sodium hypochlorite were effective at completely removing intraradicular E. faecalis biofilms. Conventional syringe irrigation with sterile saline solution was only partially effective at removing the biofilms.

The isolation of bifidobacteria from occlusal carious lesions in children and adults.

The aim of this study was to enumerate and identify bifidobacteria from occlusal carious lesions in permanent and deciduous teeth. Samples of infected dentine were obtained from 24 active occlusal lesions in deciduous teeth and from 15 occlusal lesions in permanent teeth. Plaque samples from sound occlusal surfaces of 12 caries-free adults and 12 children were also obtained. The bifidobacterial strains were isolated in mupirocin-containing selective media, Gram-stained and subcultured for identification. Total bacterial counts were determined using fastidious anaerobic agar, and isolates were identified using genus-specific PCR primers and were confirmed by 16S rRNA sequencing. Bifidobacteria were isolated from 13 of the 15 occlusal lesions in the adults and formed 5.09 +/- 2.11% of the total cultivable flora. In the children, bifidobacteria were isolated from 16 of the 24 occlusal lesions and formed 7.4 +/- 2.6% of the total flora. No bifidobacteria were isolated from the occlusal surfaces of caries-free adults or children. A total of 424 bifidobacteria were identified and these were Bifidobacteriumdentium, Parascardovia denticolens, Scardoviainopicata, Bifidobacterium longum, Scardovia genomosp. C1 and Bifidobacterium breve. B. dentium was present in 14 out of the 16 bifidobacteria-positive samples from the lesions on the deciduous teeth and in 7 out of the 13 positive lesions in adults (p = 0.04). The present data suggest that bifidobacteria may play a role in the progression of occlusal caries lesions in both children and adults.

Antimicrobial properties of dentine bonding agents determined using in vitro and ex vivo methods.

AIMS: To determine the antimicrobial properties of a selection of dentine bonding agents [DBAs] using the disc diffusion and direct contact methods and an ex vivo method using extracted carious permanent molar teeth. METHODS: DBAs (n=15) were tested using Streptococcus mutans UA159 in disc diffusion and direct contact methods. In the ex vivo study 6 DBAs were selected and pre- and post-treatment samples of carious dentine (n< or =12) were taken. Samples were also taken post-acid-etching. The number of microorganisms in dentine sample was determined and compared. RESULTS: The inhibition zones and percent growth inhibition were related to the pH of the culture medium containing the DBA (p<0.01). Clearfill Protect Bond exhibited the greatest bacterial killing followed by ibond (99.8%+/-0.08 and 98.2+/-1.4, respectively). The phosphoric acid etchant alone resulted in an 83% killing. The in vitro tests results did not correlate. The ex vivo killing reflected the percent growth inhibition observed in the direct contact method. CONCLUSION: A guide to the potential antimicrobial activity of a DBA may be gained from an assessment of its pH when added to bacteriological culture medium. The direct contact method gives a better reflection of the killing of bacteria in infected dentine than the disk diffusion method. Killing in the ex vivo model gives a more realistic and more reliable method for determining the antibacterial activity of a given DBA and that comparisons of the relative inhibitory activity of DBAs should be tested using this ex vivo model.

Association between Bifidobacteriaceae and the clinical severity of root caries lesions.

BACKGROUND/AIMS: The isolation of members of the family Bifidobacteriaceae (bifids) from oral samples has been sporadic and a recent cloning study has suggested that they are not detectable in root caries lesions. METHODS: To better understand the presence of bifids in root caries we obtained clinical samples (15 of each) from sound exposed root surfaces, leathery remineralizing root lesions, and soft active root lesions. We investigated each for the presence of bifids using a mupirocin-containing selective medium and identified the isolates using 16S recombinant RNA sequencing. RESULTS: The proportion of bifids, as a percentage of the total anaerobic count, was significantly related to the clinical status of the sites sampled, being 7.88 +/- 1.93 in the infected dentine from soft lesions, 1.61 +/- 0.91 in leathery lesions, and 0.05 +/- 0.39 in plaque from sound exposed root surfaces. Bifids were isolated from all soft lesions, 13 of 15 leathery lesions, and five of the plaque samples. Bifidobacterium dentium was isolated from four of the plaque samples, from 13 samples from leathery lesions, and from 12 of the 15 samples of infected dentine from the soft active lesions. Parascardovia denticolens and Scardovia genomospecies C1 were each isolated from samples associated with all three clinical conditions whereas Scardovia inopicata and Bifidobacterium subtile were both isolated from the infected dentine of the leathery and soft lesions. Bifidobacterium breve was isolated from the infected dentine of soft root caries lesions. CONCLUSION: Bifids may be routinely isolated from root caries lesions using appropriate cultural methods.

The predominant cultivable Veillonella spp. of the tongue of healthy adults identified using rpoB sequencing.

The predominant Veillonella spp. were isolated from the dorsum surface of the tongues of 11 healthy adults and identified to species level using rpoB sequencing because 16S ribosomal RNA sequence analysis does not reliably differentiate between all members of this genus. In all, 253 isolates were identified and the mean proportion (+/- SE) of Veillonella spp. per sample was 16.2 (+/- 3.6) with a range of 3.0% to 36.3% of the total anaerobic colony count. The predominant species were Veillonella atypica (10/11), Veillonella dispar (9/11) and Veillonella rogosae (8/11) because they were isolated from the majority of subjects. Veillonella parvula was isolated from only one subject while Veillonella dentocariosi and Veillonella montpelleriensis were not isolated from any subject.

Diversity of Veillonella spp. from sound and carious sites in children.

Detailed data on the distribution of Veillonella in caries-free and caries-active subjects are scarce. We hypothesized that the diversity of the genus would be lower in caries lesions than in plaque from caries-free individuals. The proportions of Veillonella were not significantly different in the two groups. All isolates (n = 1308) were genotyped by REP-PCR, and different genotypes (n = 170) were identified by 16S rRNA, dnaK, and rpoB sequencing. V. parvula, V. dispar, and V. atypica were in both groups, V. denticariosi only in caries lesions, and V. rogosae only from the caries-free individuals (p < 0.009). Lesions were more likely to harbor a single predominant species (p = 0.0018). The mean number of genotypes in the lesions was less than in the fissure (p < 0.001) or buccal (p = 0.011) sites. The Veillonella from caries-free sites were more diverse than those from caries lesions, and may be related to the acidic environment of caries lesions.

Assessment of the ozone-mediated killing of bacteria in infected dentine associated with non-cavitated occlusal carious lesions.

The ability of ozone to kill micro-organisms associated with non-cavitated occlusal caries was investigated. The occlusal surfaces were treated with ozone (n = 53) or air (n = 49) for 40 s, and the underlying infected dentine was exposed. There was no significant difference between the number of bacteria recovered from the ozone-treated and the control sites (p > 0.1). Treatment of the exposed dentine with ozone resulted in a just significant (p = 0.044) reduction in bacterial counts. Ozone treatment of non-cavitated occlusal lesions for 40 s failed to significantly reduce the numbers of viable bacteria in infected dentine beneath the demineralized enamel.

The potential for sodium hexametaphosphate (SHMP) found in common children drinks to limit acid production in the oral biofilm.

OBJECTIVE: Sodium hexametaphosphate (SHMP) is a widely used industrial preservative commonly found in children's drinks. In this paper we examined the effect of SHMP incorporated into children's drinks on acid production by the oral biofilm by monitoring salivary concentrations of lactic acid. MATERIALS AND METHOD: Twelve healthy adult subjects with an average age 36 years (range 26-54 years) consumed 10 ml from four children's beverages (Coca Cola and three types of Sunny Delight supplemented with SHMP) and a standard solution of sucrose. Saliva was collected at intervals following exposure of the oral biofilm to the drinks and the clearance of carbohydrates and the appearance of lactate was measured using standard enzymatic techniques. RESULTS: All the carbohydrates derived from the drinks were cleared from saliva within 15 min of consumption. Comparison of two drinks [Sunny D Normal and Sunny C] with the same carbohydrate, but different SHMP concentrations suggested that SHMP in these beverages had no significant effect on acid production. CONCLUSIONS: In this clinical study the role of SHMP, incorporated in common beverages, did not inhibit acid production from carbohydrates.

Microbiological assessment of occlusal brown-spot lesions in primary molars.

Conventional cultural methods were used to compare the plaque flora and the level of infection of the dentine underlying 51 occlusal brown-spot lesions and 21 sound occlusal sites on the primary dentition. Freshly extracted primary molar teeth were used, and occlusal brown-spot lesions and sound occlusal sites were identified using laser fluorescence (LF) and clinical visual methods. A standardized plaque sample was taken from each site, and an LF score was recorded for one discrete site per tooth. The teeth were carefully opened at each predetermined site to determine the clinical status of the underlying dentine, and samples were collected using a sterile bur. The microbiota of the plaque and dentine samples were enumerated and identified. The mean LF scores for the sound sites and brown-spot lesions were 1.2 and 30.5 (p < 0.001), and all the sound sites exhibited hard sound dentine, but 6 out of 51 brown spots exhibited softened dentine. Overall there was no significant (p > 0.1) difference between the level of infection of the dentine of the sound and brown-spot sites, although some sites in the brown-spot lesions yielded high numbers of bacteria. However, the numbers of bacteria as log10(CFU per sample + 1) +/- SE recovered from the plaque above the brown-spot lesions were significantly greater than above the sound sites, i.e. 2.89 +/- 0.24 and 0.89 +/- 0.33, respectively. These data indicate that brown-spot lesions may be more plaque retentive than sound sites and that they are either arrested or arresting lesions, which may require preventive intervention.