Infrared fluorescence measurements – The influence of calibration frequency on longitudinal in vitro measurements with KaVo DIAGNOdentTM

With today's widespread use of fluoride, the nature of cavities has changed. Harder, and therefore more resistant enamel can many times conceal subsurface decay and the caries disease progresses, in many cases, for a prolonged period with low activity and slow progression. The change in pattern of the caries disease calls for a shift in treatment philosophy; the original maxim of ‘extension for prevention’ has been eschewed for a minimal intervention approach, although this approach is only effective if caries is diagnosed at an early stage. Incorrect diagnosis results in incorrect treatment decisions. In the current age of lower overall prevalence of decay and slow disease progression, the potential risk of unnecessary restorations is greater than the risk of missing early decay. As an adjunct to conventional caries diagnostic methods such as visual inspection and bitewing radiography, a need for objective quantitative detection methods is of high importance. KaVo DIAGNOdent^TM (KaVo Dental, Biberach, Germany) is a laser fluorescence device developed for caries detection and quantification as an adjunct to visual inspection and radiographic examination. The aim of this in vitro study was to investigate the stability of the instrument for longitudinal measurements. The study was carried out in two subsequent parts where measurements were performed in two series, with and without calibration. The material in Part I of the study comprised 30 extracted teeth with various stages of carious lesions measured with one DIAGNOdent^TM device. In Part II, two devices were used to determine their unanimity and measurements were performed on six fluorescence standards in order to minimize false positive readings. The first series in Part I, with only one initial calibration, showed a significant change over time: a linear trend with drifting towards lower readings (P < 0.001). In the second series, with frequent calibrations, no significant linear trend over time could be demonstrated (P = 0.09). Clinically relevant differences in mean value between the series of measurements were seen over time (without frequent calibration, 7.64; with frequent calibration 8.57). The mean value of readings from the series with frequent calibrations was approximately 1 unit higher throughout the study, and single observations were 1–6 units higher. Results from Part II showed a significant systematic over‐time difference between the factor ‘without’ and ‘with’ calibration (P = 0.0023) independent of which device that was used (P = 0.67). There was no significant difference between the devices, DDI and DDII (P = 0.14). The interaction, time × calibration, was significant (P < 0.000) with stable readings over time in the period ‘with calibration’, while the readings in the period ‘without calibration’ was drifting towards lower readings from day 1 and forward. From this in vitro study it was concluded that frequent calibration of DIAGNOdent^TM should be performed in order to obtain comparable data for longitudinal monitoring.     

Molecular analysis of age‐related changes of Streptococcus anginosus group and Streptococcus mitis in saliva

The purpose of this study was to survey the prevalence of streptococcal species, especially Streptococcus anginosus (which has been reported to be associated with cancer in the upper digestive tract), Streptococcus constellatus, and Streptococcus intermedius in the saliva of different age groups. A sequence analysis of 16S rDNA was performed and DNA quantified using real‐time polymerase chain reaction. The S. anginosus level increased with age, whereas the levels of S. constellatus and S. intermedius did not change. Streptococcus mitis was the predominant species in the saliva of all the age groups but, unlike the S. anginosus, the proportion of S. mitis in the salivary bacteria decreased with age. The increase in S. anginosus with age should be carefully monitored because of its association with diseases, including cancer.     

Severe acute respiratory syndrome coronavirus 2 infection in asymptomatic pediatric dental patients

BackgroundChildren with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are typically asymptomatic but contagious. The authors investigated the positivity rate of asymptomatic SARS-CoV-2 infection in pediatric dental patients.MethodsThe authors reviewed consecutive charts of children younger than 18 years scheduled for elective dental procedures from April 1, 2020, through August 1, 2020. All patients were screened for signs and symptoms of SARS-CoV-2 infection. Asymptomatic patients scheduled for dental procedures underwent polymerase chain reaction (PCR) testing for SARS-CoV-2. Sociodemographic characteristics were abstracted, and positivity rates were calculated. Variables for patients who were SARS-CoV-2 positive and SARS-CoV-2 negative were compared using Fisher exact and Mann-Whitney U tests.ResultsThe sample size was 921. The median age was 6 years, and 50.9% were boys. The overall SARS-CoV-2 positivity rate was 2.3%. Age, insurance status, medical history, and dental diagnosis were comparable in patients who were SARS-CoV-2 positive and SARS-CoV-2 negative. Positivity rates were statistically higher for Hispanic or Latinx patients than other groups (P = .038).ConclusionsAlthough the yield of testing was low, the systematic evaluation of asymptomatic pediatric dental cases via PCR resulted in the identification of SARS-CoV-2 carriers who could have been infectious. In this study, Hispanics or Latinx had a higher positivity rate than other demographic groups.Practical ImplicationsPCR testing for SARS-CoV-2 of asymptomatic patients in pediatric dentistry adds value to the use of screening questionnaires for the identification of infected people who could be contagious.     

The influence of zero-value subtraction on the performance of two laser fluorescence devices for detecting occlusal caries in vitro

BACKGROUND: The aim of this study was to evaluate the influence of zero-value subtraction on the performance of two laser fluorescence (LF) devices developed to detect occlusal caries. METHODS: The authors selected 119 permanent molars. Two examiners assessed three areas (cuspal, middle and cervical) of both mesial and distal portions of the buccal surface and one occlusal site using an LF device and an LF pen. For each tooth, the authors subtracted the value measured in the cuspal, middle and cervical areas in the buccal surface from the value measured in the respective occlusal site. RESULTS: The authors observed differences among the readings for both devices in the cuspal, middle and cervical areas in the buccal surface as well as differences for both devices with and without the zero-value subtraction in the occlusal surface. When the authors did not perform the zero-value subtraction, they found statistically significant differences for sensitivity and accuracy for the LF device. When this was done with the LF pen, specificity increased and sensitivity decreased significantly. CONCLUSIONS: For the LF device, the zero-value subtraction decreased the sensitivity. For this reason, the authors concluded that clinicians can obtain measures with the LF device effectively without using zero-value subtraction. For the LF pen, however, the absence of the zero-value subtraction changed both the sensitivity and specificity, and so the authors concluded that clinicians should not eliminate this step from the procedure. CLINICAL IMPLICATIONS: When using the LF device, clinicians might not need to perform the zero-value subtraction; however, for the LF pen, clinicians should do so.     

Endpoint quantitative PCR assays for Bacteroides forsythus,Porphyromonas gingivalis,and Actinobacillus actinomycetemcomitans

BACKGROUND: Conventional polymerase chain reaction (PCR) assays for periodontal pathogens are so sensitive that they detect infections of no clinical significance. Quantitative PCR (qPCR) may provide a solution to this problem. However, most qPCR systems require expensive real-time thermal cyclers. OBJECTIVE: Our goal was to develop qPCR assays which would allow endpoint quantification. MATERIALS AND METHODS: 16S rRNA primers for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans were adapted to the Amplifluor qPCR system, which incorporates fluorescein into the PCR product so that endpoint fluorescence is proportional to the original amount of template. DNA dilutions representing known numbers of cells were used as standard curves. Pooled subgingival plaques from the four deepest pockets of 21 severe adult periodontitis patients were assayed. Buccal molar supragingival plaque from 35 dental students provided healthy controls. Endpoint fluorescence was measured with a microplate reader. RESULTS: Optimized standard curves were linear in log-log or semilog fits over a range of 100-10(6) cells. Countable B. forsythus was present in all patients, with counts (as logs) from 2.4 to 7.3 (mean = 5.0), and 11 controls with counts from 2.1 to 4.5 (mean = 3.0). P. gingivalis was present in 11 patients and no controls, with counts from 2.2 to 4.7 (mean = 3.2). A. actinomycetemcomitans was present in two patients, with counts of 1.5 and 3.5. CONCLUSIONS: Amplifluor qPCR assays discriminated between plaque samples differing by one log or more, allowing major infections to be distinguished from minor ones. This approach allows high-throughput qPCR of plaque samples, using equipment available to many laboratories.     

Alkali‐resistant bacteria in root canal systems

The aim of this study was to isolate and identify alkali‐resistant bacteria from the dentin of infected root canals. Bacteria from homogenized dentin powder made up from infected root canal walls from human teeth were cultured on buffer‐enriched Brain Heart Infusion agar supplemented with 4% sheep blood (BHI‐blood agar), adjusted to pH 7.0, 9.0 or 10.0. Incubation took place for 7 days at 37°C in an anaerobic glove box. Bacterial strains selected according to colony and morphology were subcultured in buffer‐enriched BHI broth adjusted to pH 9.0, 10.0 or 11.0 to confirm their growth as alkali‐resistant bacteria. Polymerase chain reaction amplification using specific primer sets and 16S rDNA sequence analysis was performed for identification of alkali‐resistant isolates. In the present study, 37 teeth extracted from 37 patients were used for preparation of the dentin powder samples. Bacteria were detected in 25 samples when standard BHI‐blood agars (pH 7.0) were used. Of these, 29 strains from 15 samples were alkali resistant, 25 strains growing at pH 9.0 and 4 at pH 10.0. The alkali‐resistant strains included Enterococcus faecium (10 strains) and Enterococcus faecalis (2 strains), Enterobacter cancerogenus (1 strains), Fusobacterium nucleatum (1 strains), Klebsiella ornithinolytica (2 strains), Lactobacillus rhamnosus (2 strains), Streptococcus anginosus (2 strains), Streptococcus constellatus (3 strains), and Streptococcus mitis (2 strains). Three strains were also identified as bacteria of genus Firmicutes or Staphylococcus at the genus level. The present study showed that many bacterial species in infected root canal dentin were alkali‐resistant at pH 9.0 and/or pH 10.0, and belonged mainly to the genus Enterococcus.     

Molecular evaluation of residual endodontic microorganisms after instrumentation, irrigation and medication with either calcium hydroxide or Septomixine

BACKGROUND AND OBJECTIVE: The correct choice of antimicrobial agents as inter-appointment medicaments is as important as the instrumentation and irrigation to remove pathogens from infected root canals. Calcium hydroxide [Ca(OH)2] and framycetin sulfate (Septomixine) are common endodontic medicaments. Therefore, we evaluated the efficacy of either calcium hydroxide or Septomixine in eliminating residual intra-canal bacteria, particularly Actinomyces spp., during inter-appointment interval in endodontic therapy using molecular methods. METHODS: A total of 31 single-rooted teeth with primary root canal infections were studied immediately after opening the canals and subsequently after instrumentation, irrigation with sterile saline and 1-week medication with either Ca(OH)2 (n = 25) or Septomixine (n = 6). Whole bacterial genomic DNA was isolated directly from samples and PCR with universal primers performed to detect total intra-canal bacteria. The variable regions of 16S rDNA of bacteria were amplified and labeled with digoxigenin for further hybridization to detect Actinomyces spp. A total of seven oligonucleotide probes specific for A. bovis, A. gerencseriae, A. israelii, A. meyeri, catalase-negative A. naeslundii (genospecies 1 and 2), catalase-positive A. naeslundii genospecies 2 and A. odontolyticus were used to detect Actinomyces spp. in 22 of 31 medicated root canals [Ca(OH)2: n = 17; Septomixine: n = 5]. RESULTS: The PCR results showed that 25 of 31 examined canals were positively detected with residual microorganisms after instrumentation, irrigation with sterile saline and 1-week medication with either Ca(OH)2 (n = 20) or Septomixine (n = 5). Thus, only six canals [Ca(OH)2: n = 5, Septomixine: n = 1] were aseptic after treatment. Hybridization results showed higher detection frequency of both A. odontolyticus and A. gerencseriae after treatment. Significant correlation was found between exposed pulp before treatment and positive detection of Actinomyces spp., particularly A. odontolyticus on the second visit (P < 0.05). CONCLUSION: The conventional, 1-week medication of either Ca(OH)2 or Septomixine in endodontic therapy may not effectively inhibit residual bacterial growth in all root canals during inter-appointment intervals. Further investigations using, for instance quantitative real-time PCR analyses, are required to substantiate the present findings.     

Prevalence of Porphyromonas gingivalis in relation to periodontal status assessed by real‐time PCR

Many studies have examined the presence of Porphyromonas gingivalis in periodontal pockets. However, monitoring the number of bacterial cells is difficult. In this study, we performed quantitative analyses of P. gingivalis to clarify the relationship between the numbers of this organism and periodontal status. Using the TaqMan real‐time PCR system, we found a significant positive correlation (P < 0.0001) between the number of P. gingivalis and pocket depth. The slope of the regression line indicated that for every 1‐mm increase in pocket depth, the number of P. gingivalis increased 10‐ fold. There was also a significant reduction (P < 0.01) in the numbers of P. gingivalis before and after treatment. These results suggest that the absolute and relative numbers of P. gingivalis are closely associated with periodontal status, and that quantitative analysis of this organism is important for the evaluation of periodontal therapy.