University of Iowa

Iowa Research Online

Theses and Dissertations

2013

The association between CarieScan Pro readings and histologic depth of caries in non cavitated occlusal lesion in vitro
Joshua Eric Cohen
University of Iowa

Copyright 2013 Joshua Eric Cohen This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/2463 Recommended Citation
Cohen, Joshua Eric. "The association between CarieScan Pro readings and histologic depth of caries in non cavitated occlusal lesion in vitro." thesis, University of Iowa, 2013. http://ir.uiowa.edu/etd/2463.

Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Other Dentistry Commons

THE ASSOCIATION BETWEEN CARIESCAN PRO READINGS AND HISTOLOGIC DEPTH OF CARIES IN NON CAVITATED OCCLUSAL LESION IN VITRO

by Joshua Eric Cohen

A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Operative Dentistry in the Graduate College of The University of Iowa May 2013 Thesis Supervisor: Associate Professor Justine L. Kolker

Copyright by JOSHUA ERIC COHEN 2013 All Rights Reserved

Graduate College The University of Iowa Iowa City, Iowa

CERTIFICATE OF APPROVAL _______________________ MASTER'S THESIS _______________ This is to certify that the Master's thesis of Joshua Eric Cohen has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Operative Dentistry at the May 2013 graduation. Thesis Committee: ___________________________________ Justine L. Kolker, Thesis Supervisor ___________________________________ Gerald E. Denehy ___________________________________ Fang Qian

Dedicated to the loving memory of my mother, Linda Cohen (1956-2010) I Love You Mom

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ACKNOWLEDGMENTS I would like to sincerely thank my thesis committee members: Dr. Justine L. Kolker, Dr. Gerald E. Denehy, and Dr. Fang Qian for their guidance and encouragement. I would also like to thank Dr. James Wefel who allowed me to get a glimpse of his infinite wisdom in Cariology. A special thank you is in order to Dr. Christopher Longbottom, Dr. Woosung Sohn, Dr. Gail Douglas, Dr. Marcos A. Vargas, Dr. Rodrigo R. Maia, Jeffrey Harless, and Maggie Hogan for taking time out of their busy schedules to offer much needed technical expertise. I am especially thankful to my wife Alison and my daughters Emily, Madeline, and Sophia for their unwavering love and support. I simply could not have done this without you.

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TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER I INTRODUCTION. .........................................................................................1 Purpose .............................................................................................................3 Study Aims .......................................................................................................4 CHAPTER II LITERATURE REVIEW .............................................................................5 Introduction.......................................................................................................5 History of Dental Caries .............................................................................6 Caries Prevalence Post Water Fluoridation ................................................6 Current Dental Diagnostic Techniques .......................................................7 Mechanisms Involved in Caries Process ..........................................................7 Bacterial Involvement ..................................................................................7 Sugar Consumption .....................................................................................8 Oral Cavity Environment .............................................................................8 Demineralization/Remineralization .............................................................9 Caries Presentation .........................................................................................10 Non-Cavitated Lesions ..............................................................................10 Cavitated Lesions.......................................................................................11 Closed Lesions ...........................................................................................12 Caries Activity ...........................................................................................12 Caries Detection Techniques ..........................................................................13 Visual Detection ........................................................................................13 International Caries Detection and Assessment System (ICDAS) ............14 Nyvads System .........................................................................................14 Visual/Tactile Detection ............................................................................18 Radiographic Detection .............................................................................22 Caries Detection Devices ................................................................................28 Fiber Optic Transillumination (FOTI) .......................................................28 DIFOTI ......................................................................................................32 Laser Fluorescence ....................................................................................33 Quantitative Laser Fluorescence (QLF) ....................................................34 Light Induced Fluorescence (DIAGNOdent) ............................................37 Electrical Conductance ..............................................................................41 Electronic Caries Monitor (ECM) .............................................................43 CarieScan PRO ..........................................................................................47 CHAPTER III MATERIALS AND METHODS ..............................................................50 Tooth Selection ...............................................................................................50 Digital Macro Photographs of Occlusal Surfaces ...........................................50 Scoring Occlusal Lesions ...............................................................................51 ICDAS II Criteria Code .............................................................................52 Caries Analysis Uisng CarieScan PRO ..........................................................53 Charging the CarieScan PRO ....................................................................53 System Testing...........................................................................................53 iv

Scoring Teeth with the CarieScan PRO.....................................................53 Tooth Sectioning .............................................................................................55 Tooth Preparation for Sectioning...............................................................55 Mounting Teeth for Sectioning ..................................................................56 Sectioning ..................................................................................................56 Removing Sections ....................................................................................57 Polarized Light Microscopy ...........................................................................57 Histologic Examination ..................................................................................58 Ranked Scale .............................................................................................58 Statistical Analysis..........................................................................................59 Intra- and Inter-Rater Reliability for Visual Inspection ............................59 Intra-Rater Reliabilityof Examination with the CarieScan PRO ...............60 Intra- and Inter-Rater Reliability of Histologic Examination ....................60 Association Between Visual Inspection and Histologic Findings .............60 Association Between CarieScan PRO and Histologic Findings ................61 Association Between Visual Inspection and CarieScan PRO ...................61 Sensitivity and Specificity for ICDAS ......................................................61 Sensitivity and Specificity for the CarieScan PRO ...................................63 Pilot Study ......................................................................................................65 Hypotheses ......................................................................................................65 Operational Definitions ..................................................................................65 CHAPTER IV RESULTS ..................................................................................................91 Evaluations of Intra- and Inter-Observer Reliability for Measurements ........91 Intra-Observer Reliability for ICDAS Scores............................................91 Inter-Observer Reliability for ICDAS Scores............................................91 Intra-Observer Reliability for CarieScan PRO Scores ..............................92 Intra-Observer Reliability for Histologic Scores .......................................93 Inter-Observer Reliability for Histologic Scores .......................................93 Evaluations of the Associations Between Histologic Consensus, ICDAS Consensus, and CarieScan Pro Mean ................................................94 Association of Histologic Consensus with ICDAS Consensus .................94 Association of Histologic Consensus with CarieScan PRO Mean ............94 Association of CarieScan PRO Mean with ICDAS Consensus.................95 Sensitivity and Specificity ..............................................................................97 Sensitivity and Specificity of ICDAS ........................................................97 Sensitivity and Specificity of CarieScan PRO...........................................98 CHAPTER V DISCUSSION ...........................................................................................107 REFERENCES ................................................................................................................117

LIST OF TABLES Table 1. 2. 3. 4. 5. 6. 7. 8 9. 10. 11. 12. 13. 14. 15. 16. 17.
ICDAS vs Histo I. ....................................................................................................62 ICDAS vs Histo I Interpreted....................................................................................62 ICDAS vs Histo II. ...................................................................................................63 CarieScan vs Histo I. ...............................................................................................63 C.S. vs Histo I Interpreted. .......................................................................................64 CarieScan vs Histo II. ..............................................................................................64 ICDAS vs Histo I .....................................................................................................97 ICDAS vs Histo II. ..................................................................................................98 CarieScan vs Histo I. ...............................................................................................98 CarieScan vs Histo II. ..............................................................................................99

Nyvad et als description of diagnostic criteria from a 1999 paper in Caries Research..................................................................................................................100 Criteria used for the visual, FOTI and radiographic examinations in Crtes et als 2000 paper in Caries Research ........................................................................101 Descriptive statistics of mean differences between the first and second measurements with the CarieScan PRO ................................................................102 Associations of ICDAS levels with histologic consensus categories (N=95) .......103 Associations of CarieScan levels with histologic consensus categories (N=95) ...................................................................................................................104 Associations of CarieScan levels (0-50, 51-100) with ICDAS levels (N=95). .....105 Associations of CarieScan levels (0-30, 31-100) with ICDAS levels (N=95). .....106

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LIST OF FIGURES Figure 1. 2. 3. 4. 5. 6. 7. 8. 9. Tooth Mounted for Photographs ...............................................................................67 Microsoft PowerPoint image of photographed teeth. Wet image on the left. Dry image on the right. Red circle denotes the area of interest ................................68 Photograph of the Excel spreadsheet used to randomize the photographed lesions to prevent any bias during second evaluation ...............................................69 ICDAS Codes 0 - 4. .................................................................................................70 CarieScan PRO fully charged in cradle per manufacturer's specifications. .............71 Sensor collar in place calibrating the CarieScan PRO per manufacturer's specifications. ...........................................................................................................72 Proper placement of the CarieScan PRO sensor.......................................................73 CarieScan color pyramid and corresponding numbers. ............................................74 With the lip hook in contact with the tooth and wrapped in a gauze saturated in artificial saliva, the CarieScan PRO sensor is placed on the site of interest and a score is recorded. .............................................................................................75 Placing buccal (red) and lingual (green) marks to designate location of the lesion prior to covering occlusal surface with resin. ................................................76 Etching the occlusal surface prior to placing resin. ..................................................77 Frosted appearance in the enamel after acid etching. ...............................................78 Placing an adhesive prior to resin placement on the occlusal surface. .....................79 Placing composite resin on the occlusal surface. ......................................................80 Series 1000 Deluxe Hard Tissue Microtome. ...........................................................81 One of four cuts in the tooth to get three sections. ...................................................82 Mounting ring ready to be placed in microtome for sectioning. The tooth was mounted high enough in the wax to expose the CEJ. ...............................................83 Serial sections in a buccal/lingual direction through the site of interest which was identified by the paint. .......................................................................................84 Sections cut into the pulp chamber so that the extent of caries into dentin can be properly assessed..................................................................................................85 Separating the second of three sections using the Interproximal Carver (IPC). .......86 vii

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

Serial sections prepared for histologic exam with Polarized Light Microscopy (PLM). Sections placed on the slide in the order they were separated. The red paint can be seen on the buccal surfaces of the middle and left sections. ..........87 Looking at sections using an Olympus BH-2 Polarized Light Microscope. ............88 Image taken from PowerPoint document used to score histologic lesions. The images from left to right correspond with the sections on the slide as seen in Figure 21. ..................................................................................................................89 Examples of histologic scoring using the Crtes ranked scale. ................................90

22. 23.

24.

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CHAPTER I INTRODUCTION

Since the implementation of wide spread water fluoridation and other fluoride modalities in the last half century, the incidence and prevalence of dental caries in the United States has decreased. Despite this decrease, dental disease is still a problem today. Due to the complexity of the disease process, it is often difficult to diagnose dental caries. In many circumstances, the presence of fluoride can help hide occlusal caries by aiding in the initial mineralization and remineralization of enamel, making it difficult for providers to effectively detect, manage, and treat the disease at the earliest possible stage. Studies have shown inconsistencies in the ability for dentists to uniformly detect dental caries. Dentists generally rely on a few methods for diagnosis, including visual and/or tactile, and radiographic. While radiography is a valuable diagnostic tool, its usefulness at diagnosing occlusal caries is limited. By the time occlusal caries can be detected radiographically, the disease process is already quite advanced minimizing the opportunity for providers to treat at a minimally invasive level. Visual and tactile methods of caries detection are often reported to be highly subjective (Pereira, Eggertsson et al. 2009). Clinicians often have different perceptions of what is considered soft tooth structure or what characteristics are indicative of tooth decay on a specific tooth surface. This inability for dental professionals to consistently detect caries using visual and tactile methods calls for a more objective method which could potentially quantify levels of dental disease. Additionally, a more quantifiable system of measuring decay would prove extremely valuable in future research because higher levels of studies, such as systematic reviews and meta-analyses, would be more feasible with the ability to compare scientific literature based on a quantifiable system.

To date, there have been multiple studies which have looked at various diagnostic techniques/systems and have measured their correlation to disease presence (Bader JD, Shugars DA et al. 2001, Bader JD, Shugars DA et al. 2002). Despite these studies, there has been little success in predictably correlating disease progression with clinical detection. Some systems such as the International Caries Detection and Assessment System (ICDAS), while proving to be a good diagnostic tool, require extensive calibration and training, and still have yet to show an improved ability to predict disease progression. The most exact way of measuring the spread of caries is to examine extracted teeth histologically and compare the findings with predicted outcomes of various diagnostic modalities. This experimentation has been performed with multiple diagnostic tools including: Digital Image Fiber Optic Transillumination, Fiber Optic Transillumination (DIFOTI, FOTI); Quantitative Light Fluorescence (QLF); Laser Fluorescence; and Electrical Conductance Measurement (ECM) to name a few. In general, these studies have found these diagnostic tools to have good sensitivity in that they are good at locating dental caries. The problem is that they tend to lack good specificity in that they identify an undesirable level of false positive readings, making them less useful in the practice of minimally invasive dentistry. Relatively new to the market is the CarieScan Pro, which relies on Electrical Conductance. The CarieScan PRO system uses Alternating Current (AC) Electrical Impedance, compared to its predecessor ECM, which used Direct Current (DC) as its energy source, a method already deemed non-effective at caries detection (Bader JD, Shugars DA et al. 2002). By determining the association of the CarieScan PRO readings to histological lesion depth, we can potentially predict disease progression much more effectively than ever before.

Purpose

This study evaluated the association between CariesScan PRO readings and histologic involvement measured using polarized light microscopy of non-dentinal cavitated occlusal tooth surfaces classified according to ICDAS. The study described herein is one of the first of its kind with this device, and has the potential to change the way dental caries are detected, monitored, and diagnosed. A strong association between CarieScan Pro readings and histologic involvement would eventually enable clinicians to predictably determine the progression of dental disease objectively. Removing subjectivity from the diagnostic process would be groundbreaking in clinical dentistry, forever changing the way lesions are managed and monitored. From a research standpoint, the effects of future dental materials, antimicrobials, dental fluoride delivery systems, etc. on teeth could be measured clinically without the use of invasive procedures. As described fully in chapter 3, data for this study was collected from extracted posterior teeth presenting with non-dentinal cavitated carious lesions, which were deemed as such according to ICDAS criteria (Codes 0-4). The teeth were photographed which was used to classify the lesions. Occlusal surfaces were then examined using the CarieScan PRO device. The teeth were then sectioned and examined histologically. This study then examined the association between CarieScan PRO readings and actual histologic progression of dental caries.

Study Aims

The aim of this study is to measure the association between CarieScan PRO readings and histologic depth of caries on non-dentinal cavitated occlusal lesions in vitro. Additionally, this study aims to measure the association between ICDAS scores and histologic depth of caries on the same surfaces. The association between ICDAS scores and CarieScan PRO readings will also be measured. The CarieScan PRO manufacturer claims sensitivity and specificity greater than .90. This study intends to compare sensitivity and specificity findings with manufacturer claims.

CHAPTER II LITERATURE REVIEW

Introduction

Diagnosis of dental caries and deciding when to provide treatment is still considered very subjective and continues to be widely debated to this day. There continues to be considerable research regarding this subject matter in an attempt to better understand the caries progression process and how it can be prevented. Researchers, in an attempt to standardize the methods with which they collect data, have used the World Health Organizations criteria for diagnosing caries (World Health Organization. Expert Committee on Mental Health. 1962). Unfortunately, using this tool limits caries diagnosis to teeth that are cavitated, a clinical sign that manifests itself after extensive progression in the disease process. Other classification systems have come into use in order to detect dental caries earlier. Unfortunately, there is inadequate evidence to support that these systems can be used as universally as the WHO system. To complicate the matter, dental caries have become increasingly more difficult to diagnose since the widespread use of fluoride. Non-cavitated dentinal lesions are far more prevalent as a result of little to no demineralization on occlusal surfaces making detection much more difficult (Sawle RF, Andlaw RJ 1988, Weerheijm KL, van Amerongen WE et al. 1989). Even when occlusal caries are detected, there is debate as to the extent of the caries progression and whether or not it should be treated. The ability to quantitatively measure the progression of caries would be a useful clinical tool in objectively identifying the extent of the disease process. Additionally, a predictable quantitative measure could improve caries research by ensuring uniformity in lesion progression without relying on inter-rater reliability. The following literature review will address the current knowledge of caries detection and the respective techniques used in this decision making process.

History of Dental Caries

Dental caries is an ancient disease that can be traced back as far as the fifth century. It is widely believed that the origin of dental caries may have occurred shortly after agriculture replaced hunting and gathering as the primary source of food. Examination of skulls in Britain suggests that the moderate caries experience found in the Anglo-Saxon period (fifth to seventh centuries) had changed little by the end of the Middle Ages, approximately the year 1500(Moore, Corbett 1971, Moore, Corbett 1973). By the eighteenth century sugar was much more available as was the prevalence of food refinement. By the end of the nineteenth century, dental caries was established as a worldwide endemic disease in developed countries.

Caries Prevalence Post Water Fluoridation

The overall prevalence of dental caries has drastically declined over the last thirty to forty years (Alwas-Danowska HM, Plasschaert AJ et al. 2002). Due to increased preventive modalities such as the emergence of fluoridated drinking water and toothpastes, there has been a dramatic decrease in smooth surface caries resulting in an increased proportion of occlusal caries as a proportion of total caries prevalence (AlwasDanowska HM, Plasschaert AJ et al. 2002). Early occlusal caries detection has become more difficult due to the absence of cavitation in and underneath fissures as a result of frequent fluoride use (Alwas-Danowska HM, Plasschaert AJ et al. 2002). Because of this fact, today there is a greater need for early occlusal caries diagnosis.

Current Dental Diagnostic Techniques

Dentists have a number of ways of detecting occlusal caries. Some of these methods include: visual, visual-tactile, radiographic, laser or light fluorescence, fiber optic transillumination, and electrical impedance (Jablonski-Momeni, Stachniss et al. 2008). With all these options at their disposal, visual, tactile, and radiographic are currently the most widely used diagnostic tools in caries detection (Adeyemi AA, Jarad FD et al. 2008).

Mechanisms Involved in Caries Process

Bacterial Involvement

Bacteria are necessary for the occurrence of dental caries regardless of any other factor. Caries cannot occur in the absence of bacteria (Emilson CG, Krasse B 1985, Loesche 1982). The primary bacteria responsible for dental caries are mutans streptococci and lactobacilli, which are naturally occurring in the oral cavity. Because of the natural occurrence of these organisms, as well as others, it is widely accepted that an imbalance in the number of these bacteria with respect to the total bacterial count plays an integral role in the development of dental caries. However, bacterial counts by themselves are a poor predictor of caries development. In order for caries to develop, an environment must be created that is rich in bacteria, has an adequate substrate (teeth and the oral cavity environment) and a food source. Caries have been described as a carbohydrate-modified bacterial infectious disease, in which a cariogenic diet selectively favors cariogenic bacteria (van Houte J, Lopman J et al. 1994).

Sugar Consumption

Similar to bacterial involvement, diet plays an integral role in the development of dental caries. This remains particularly true in regards to the intake of refined carbohydrates and sugars. When these sugars have been refined prior to consumption, they are broken down much easier in the oral cavity making them readily available for consumption by cariogenic bacteria. Sugars added to the diet seems to be the primary cause of caries, however, caries can also occur in populations whose only sugar consumption is naturally occurring. Despite the overwhelming evidence in the literature to support this claim, it is important to note that sugars are not the only food source involved in the carious process. Cooked or milled starches can be broken down to low-molecular-weight carbohydrates by the salivary enzyme amylase and thus act as a substrate for cariogenic bacteria. (Bibby BG 1975, Firestone, Schmid et al. 1984). It has been argued that a mixture of sugars and starches are more cariogenic than sugars by themselves. Conversely, foods containing high molecular weight carbohydrates when lightly cooked, (i.e. vegetables) are not considered cariogenic because they cannot be broken down completely by amylase in the mouth and therefore not an adequate food source for cariogenic bacteria(Krasse B 1982, Newbrun E, Hoover C et al. 1980).

Oral Cavity Environment

As alluded to earlier, the oral cavity is saturated with a diverse population of resident microflora. These microflora, particularly bacteria, contribute to the normal physiology of a host species in direct and indirect ways. Bacteria may colonize and therefore occupy host sites preventing the colonization of other species which may be harmful to the host. Additionally these bacteria may create microenvironments which

prove to be unfavorable for the growth and development of invading species which may be harmful to the host (Kuramitsu, Ellen 2000). Microflora is not the only component in the oral cavity that affects the development of dental caries. Saliva composition and volume are very important in creating a symbiotic relationship between host and microflora species. One of the important functions of saliva is to dilute and eliminate substances introduced into the mouth. When sugar concentrations rise in the mouth, saliva production is increased. This increase in volume will induce swallowing, thus clearing the mouth of sugar. The remaining sugar is then gradually diluted by incoming saliva. Saliva also has the ability to act as a buffer, keeping the pH of the oral cavity high enough to prevent demineralization in tooth surfaces (Fejerskov 2009).

Demineralization/Remineralization

Normal tooth enamel is predominantly mineral in content. It consists of hydroxyapatite crystals, primarily made up of calcium phosphate, tightly packed together giving it a glass like appearance. Enamel exposed to the oral environment experiences constant surface changes and modifications due to the recurring changes in pH generated by diet and plaque accumulation. During acid exposures, calcium and phosphate are precipitated out from the tightly packed hydroxyapatite lattice leading to demineralization of tooth structure. As the acid is buffered and the pH levels begin to rise, Calcium and Phosphate are re-incorporated into the Hydroxyapatite matrix causing the tooth to remineralize. These phases of demineralization and remineralization, when in balance, result in no net mineral loss, therefore preserving the structural integrity of the tooth surface. During remineralization, and in the presence of fluoride ion, small amounts of fluoride are removed from solution during crystal growth. The hydroxyapatite molecules

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substitute a hydroxyl group with fluoride, creating a fluorapatite molecule. These changes in ion content influence the physical and chemical properties of the mineral and, most importantly with respect to enamel, change its solubility (Fejerskov 2009). Fluorapatite is less soluble than hydroxyapatite rendering tooth enamel more stable during periods of acid exposure. It is this process that is desired when implementing fluoride into drinking water and toothpaste. While fluoridation has had a profound effect on the caries prevalence, it must be understood that increased fluoride concentrations in the mouth do not necessarily mean that caries will not occur. Furthermore, fluoride does not prevent the initial carious attack, which would be expected if its presence in the enamel crystal increased enamel resistance to acid dissolution, but rather the fluoride in the oral cavity acts to inhibit further demineralization of the lesion and to help promote remineralization (Burt, Eklund 2005). Because of this, dental caries presents in a much different manner than it did prior to the use of fluoride.

Caries Presentation

Non-Cavitated Lesions

Non-cavitated lesions can appear in a number of ways. Ekstrand et. al. (1995) introduced a visual ranked scale which included two types of presentations of noncavitated lesions. Ekstrand reported that non cavitated lesions can be identified by no or slight change in enamel translucency after prolonged air-drying for five seconds (Ekstrand, Ricketts et al. 1998). This is the earliest detectable presence of the caries process visually. More involved would be an opacity or discoloration to the enamel barely detectable on a wet tooth surface, but visibly noticeable upon air drying. Still more involved, they can present as distinctly opaque or discolored visibly without air

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drying of the tooth surface. Most involved, non-cavitated lesions present as localized areas of enamel breakdown in opaque or discolored enamel and/or a grayish discoloration from the underlying tooth structure. It is important to mention that not all opacities and/or discolorations are attributed to dental caries. These presentations are indicative of a lower mineral content within the enamel, however it is highly possible that these manifestations are attributed to a number of different mechanisms during enamel formation or even after tooth eruption (Fejerskov 2009). Theoretically, non-cavitated lesions can be managed by non-operative interventions. Without cavitation, there is no indication for operative intervention based on presentation alone. All non-cavitated lesions should, at the very least, be treated preventively by toothbrushing with a fluoridated toothpaste. In some cases, further preventive methods may be necessary such as fluoride varnish applications. These determinations are ultimately based on an individuals overall risk level.

Cavitated Lesions

Cavitated lesions, as one would expect, present with a cavitation in either opaque or discolored enamel, exposing underlying dentin (Ekstrand, Ricketts et al. 1998). It is generally concluded that cavitation results from the demineralization of underlying dentin causing the overlying enamel to fall upon itself resulting in cavitation. Size of cavitation, however does not always reflect the degree to which the underlying dentin has been affected. It is entirely possible for a tooth to present with fairly substantial demineralization into dentin and have only minimal cavitation.

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Closed Lesions

To understand closed lesions, one must apply the concepts of demineralization/remineralization and non-cavitated carious lesions to one another. Generally, the non-cavitated lesion only progresses to cavitation upon the complete breakdown of overlying enamel. Bjorndal describes this phenomenon occurring only after the communication of bacterial invaded enamel with hypermineralized dentin. It is after this communication that dentin begins to demineralize (Bjrndal L 2008). No serious microbial invasion takes place in the dentin as long as the highly organized enamel layer (even though being demineralized) separates the biofilm from the dentin. The bacteria are not able to penetrate through the enamel rod structure. The microbial invasion is related to the gradual structural breakdown of the enamel layer (Bjrndal L 2008). The presence of fluoride has had a major impact on the prevention of enamel breakdown as a result of: antibacterial properties; ability to prevent demineralization; ability to aid in remineralization (Burt, Eklund 2005). As a result these lesions present themselves with lesions into dentin, despite the appearance of intact enamel due to the presence of fluoride. Caries Activity

A lesion is considered to be active when the tooth undergoes mineral loss due to the metabolic activity of the biofilm adhered to the tooths surface. When biofilm activity does not result in mineral loss of tooth structure, the lesion is considered inactive. Activity may change status multiple times over the lifetime of a lesion. Nyvad et al. developed a visual tactile caries diagnostic system to determine caries activity (Nyvad, Machiulskiene et al. 1999). The philosophy behind the system is that surface characteristics of enamel are affected by biofilm activity. Therefore, when the biofilm is in a state where its activity results in mineral loss, it can be concluded that the caries

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process is active. Nyvads system focuses on surface characteristics rather than the depth of the lesion. The surface texture is an indicator of actual caries activity. The surface integrity is dependent on the presence of any cavitations or activity within the enamel. An active non-cavitated lesion is characterized as a whitish/yellowish opaque surface with a loss of luster, exhibiting a chalky or neon-white appearance (Fejerskov 2009). The surface feels rough when explored. The inactive version of this lesion conversely is shiny and feels smooth when explored. The color can vary from whitish to dark in color, however it is generally considered a non-reliable indicator. An active cavitated lesion appears soft and leathery, while an inactive lesion appears shiny and is hard on probing.

Caries Detection Techniques

Visual Detection

Visual examination is the most commonly used method for detecting caries lesions, because it is an easy technique that is routinely performed in clinical practice (Pitts NB 1993). Traditionally, visual inspection has presented with levels of high specificity, but low sensitivity and reproducibility. It is believed the levels of low reproducibility are attributed to the subjective nature of caries detection (Braga M.M., Mendes F.M. et al. 2010, Braga MM, Martignon S et al. 2010). As a measure to improve on low sensitivity and reproducibility values, several indices have been created. Two such indices discussed in this section are the International Caries Detection and Assessment System (ICDAS) and Nyvads System.

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International Caries Detection and Assessment System (ICDAS)

In an attempt to propose an internationally accepted caries detection system, an index for caries diagnosis, the ICDAS, was created in 2002 by a group of cariologists and epidemiologists, based on visual examination aided by a WHO probe (Pitts NB 2004). The teeth are cleaned and air dried for five seconds prior to examination. All examinations are performed with a dental light, a mirror, and a WHO probe as needed. Teeth are scored on an ordinal scale ranging from 0 6. The score and criteria are listed as follows: 0 No or slight change in enamel translucency after prolonged air drying (5s) 1 First visual change in enamel (after air drying or restricted to pit and fissure) 2 Distinct visual changes in enamel 3 Localized enamel breakdown in opaque or discolored enamel 4 Underlying dark shadow from dentin 5 Distinct cavity with visible dentin 6 Extensive distinct cavity with visible dentin (involving over half of a surface)

Initially, ICDAS was devised as a detection system for primary caries. Adjunct criteria have recently been devised for activity assessment.

Nyvads System

Due to the decreasing prevalence of dental caries in children and adolescents, Nyvad et. al. (1999) predicted the need for a more sensitive method of detecting caries as a result of a better understanding of the complexities involved in the dental caries process (International Conference on the Declining Prevalence of Dental Caries, Glass 1982, Marthaler, O'Mullane et al. 1996). It was concluded that the WHO method of measuring

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caries at a cavitated stage is no longer adequate in reflecting the changes in incidence of dental caries. Currently, it is better understood that the caries process is highly dynamic, and significant caries activity occurs far before the clinical manifestation of cavitation. The use of a caries diagnostic system which includes non-cavitated caries has the distinct advantage that all stages of lesion formation development of cavitation through non-cavitated stages of caries may be reflected in the recordings (Nyvad, Machiulskiene et al. 1999). The aim of Nyvads study was to describe a set of clinical diagnostic criteria differentiating between levels of caries activity in cavitated and noncavitated lesions. It was also important that they could assess the inter- and intra-rater reliability of examiners over the three year study as well as compare degrees of agreement with the commonly used WHO criteria (Table 11). Eight hundred eighty-nine, 9-14 year-old children with a high caries prevalence were selected. These children participated in a clinical caries trial and were available for repeated caries examinations for 3 consecutive years. Each year, 50 children were selected for assessment of inter- and intra-rater reliability (Nyvad, Machiulskiene et al. 1999). Two examiners independently examined each patient. Each patient was reexamined with an interval of 1-2 weeks. The percentage agreement of the caries diagnoses varied between 94.2 and 96.2%. The kappa values ranged between 0.74 and 0.85 for intra-examiner examinations and between 0.78 and 0.80 for inter-examiner examinations (Nyvad, Machiulskiene et al. 1999). The results of the study demonstrated that when the new criteria were applied to the site-specific diagnosis of caries lesions in a clinical trial, they could be reproduced by the same examiner or by another examiner with significant agreement (Nyvad, Machiulskiene et al. 1999). It was also found, as would be expected, that there were misclassifications. The majority (~80%) of these misclassifications involved disagreement between sound tooth surface and non-cavitated caries lesions (either active or inactive). Approximately 10%

16

of the misclassifications involved disagreement between non-cavitated active and noncavitated inactive lesions. Nyvads system was designed to assess activity of cavitated and non-cavitated caries lesions. In Nyvads system, one would observe a lesions surface characteristics and assess caries activity as a result of the findings. This system has presented construct and predictive validity (the different status of caries lesions can be predictive of different outcomes) concerning caries lesion activity status (Nyvad, Machiulskiene et al. 1999). Based on this system, if one characteristic presented that was consistent with active caries, that lesion was considered active. Although it was not the initial intention of the index design to do so, it was found at a later time that this index was successful in assessing the depth of lesions on primary teeth (Braga, Mendes et al. 2009). Braga et al. (2009) compared Nyvads system to ICDAS-II in conjunction with Lesion Activity Assessment (LAA) in assessing occlusal caries on primary molars in vitro. The aims of the study were to evaluate reproducibility of both systems in detecting occlusal caries, to test how well they could predict lesion depth, and to determine the correlation between the Nyvad and LAA criteria in assessing caries activity (Braga, Mendes et al. 2009). Sixty-nine primary molars were examined by two calibrated examiners. Both the Nyvad system and the ICDAS-II and LAA were used by both examiners. These findings were then compared to histologic findings, also performed by the same two examiners, of the teeth after they had been sectioned. The Spearman Coefficient was used to compare their correlation. Receiver operating characteristic (ROC) curves were used to measure cutoffs. Sensitivity, specificity, and percent agreement were calculated. Both the weighted and un-weighted kappa values for inter and intra-rater reliability were found to be very good. Due to the strong inter-examiner agreement for both visual systems and for the histological examination, consensus data were used. The sensitivity and the area under the ROC curve of the ICDAS system were significantly

17

higher than those of the Nyvad system at the D1 threshold, but not at the D2 or D3 thresholds. There were no statistically significant differences found between the two systems for specificity or percent agreement at any of the thresholds. This study concluded that there were some differences between the two systems, however they were both considered reliable in estimating the caries lesion depth on occlusal surfaces on primary teeth. Despite the strong correlation, the inability to look at plaque accumulation on extracted teeth was discussed, a key component to the Nyvad index, creating an obvious flaw to the study. It was acknowledged that the comparisons of the two systems were incomplete and that further studies were needed. Braga et. al. (2010) also compared Nyvads system with ICDAS-II LAA in vivo. The overall aim of the study was to compare the performance of the two systems in detecting and assessing caries activity of occlusal lesions. Similar to the in vitro study described earlier, two examiners examined selected tooth surfaces using both indices. One hundred sixty-four children were screened. One hundred thirty-nine children completed the examination and 763 teeth were sampled. A small subsample of teeth from this population were extracted, hemi-sectioned and examined histologically by the same two examiners. The same statistical analyses were used to examine inter and intrarater reliability, correlation, sensitivity, specificity, and accuracy as were used in the in vitro study. Again, both indices were found to have excellent inter and intra-rater reliability. It is important to note that for inter-rater reliability, the disagreements were related to non-cavitated caries lesions using both sets of criteria. Similar to the previous study, the initial stages of caries led to most of the disagreements between examiners, as expected, and, according to previous studies, accurate assessment and strong reliability demands more training and examination time (Braga, Mendes et al. 2009). It was also interesting to note that the ICDAS-LAA index scored more lesions as active compared to the Nyvad index. Additionally, it was found that neither index was able to accurately detect

18

differences between lesions in the outer and inner half of enamel. It is important to note this observation. Despite the conclusion that both indices were found to be comparable, giving high reproducibility and validity to detect and estimate overall caries lesion depth in primary teeth, there was still difficulty determining the extent of enamel only involvement (Braga MM, Martignon S et al. 2010). It is possible that this outcome would not be found in permanent dentition, and that either index may be an excellent tool in predicting caries spread in enamel. Ultimately, similar to the in vitro study, it was found that both scoring criteria were comparable. There was high reproducibility and validity to detect and estimate caries depth in primary teeth.

Visual/Tactile Detection

It has been concluded in the literature that the diagnosis of occlusal caries is indeed very difficult. In 199l Lussi looked at the validity of both clinical and diagnostic treatment decisions on occlusal pits and fissures using visual and visual/tactile methods. In this in vitro study, 61 human teeth were examined by 34 dentists. Twenty-six of these dentists were asked to examine the occlusal surfaces of extracted teeth without the use of an explorer. The remaining 8 dentists were arbitrarily assigned to use a probe to look at the same teeth. All examiners were told that the teeth were from teenagers with an average caries experience, and were given 50 seconds per tooth for examination. The examiners were responsible for diagnosing in a designated fissured area whether that fissure had (1) no caries; (2) a subsurface lesion; (3) caries confined to enamel; (4) caries beyond the dentinal-enamel junction (Lussi 1991). The examiners were also asked to assign a treatment based on their diagnosis. Their treatment options were: (1) no treatment; (2) fissure sealing; (3) preventive resin restoration; (4) composite or amalgam (Lussi 1991). Each participant was invited to repeat their examination after a minimum of one weeks time. Twelve dentists accepted this invitation.

19

Upon completion of the second examination, all teeth were sectioned into three slices which encompassed the lesion in question. The assessment of the sectioned area was the validating criterion for the evaluation of the percentage of teeth correctly diagnosed, and of sensitivity and specificity (Lussi 1991). For the sake of the study it was assumed that caries confined to enamel did not require a restoration. Based on this assumption, no treatment or fissure sealing was the correct treatment when there was either no caries, a subsurface lesion, or enamel caries. When there were dentinal caries, a composite or amalgam restoration was the treatment of choice. It was also assumed that a dentist planning to do a preventive resin restoration would switch to an amalgam or composite restoration once caries extension was revealed during treatment and therefore acceptable treatment decision when caries were found to be in dentin (Lussi 1991). It was found that the specificity of dentists using an explorer was slightly higher (87.4%) than the specificity of dentists who did not (82.5%). The sensitivity however, was slightly less (60.5%) with the explorer than without (65%) (Lussi 1991). These findings were not found to be significantly different, contributing to the idea that there is no net benefit in using tactile methods to diagnose caries. Additionally, there is adequate literature to support the disadvantages of probing fissures such as transfer of cariogenic bacteria and/or damage to the integrity of surface enamel (Lussi 1991). The results showed that the percentage of correctly diagnosed teeth in this study was rather low (approximately 42%). After removing the probability of correct diagnosis by chance alone, the proportion of correctly classified teeth beyond chance ranged between 21% (dentists using explorers) and 25% (dentists using a visual technique) (Lussi 1991). This difference was not found to be statistically significant. More importantly it reaffirms the idea that diagnosis of occlusal caries is difficult to diagnose. Penning et al. looked at the validity of probing for fissure caries diagnosis in 1992. In this in vitro study, 100 extracted molars (50 upper and 50 lower), which presented with

20

discolored fissures but no visible cavitation were selected. Each tooth was probed with a sharp sickle explorer with 500g of force. If the tooth was lifted after retracting the explorer, it was deemed to have a stick and the area was marked on the tooth with red paint. All teeth were photographed and sectioned bucco-lingually. These sections were then radiographed and examined for caries. The radiographic scores were compared with the sticks and analyzed statistically (Penning, van Amerongen et al. 1992). The results found that throughout the 100 teeth there were 1,140 probings, resulting in 41 sticks (Penning, van Amerongen et al. 1992). Upon radiographic examination, it was found that 36 of the 41 sticks were caries (true positives) with 5 being sound (false positives). Of the 1,009 non-sticking probes, 112 of them presented with caries radiographically (false negatives), while 987 of them presented as sound (true negatives). Of the 148 lesions, only 36 were marked by a sticking explorer resulting in a sensitivity of 24.3%. A false positive was found 5 out of 992 possible instances resulting in a specificity of 99.5% (Penning, van Amerongen et al. 1992). There were several limitations in this study as discussed in the paper. Some of these limitations include: the study was performed in vitro; force of probing and withdrawal on the probe, and incomplete picture of total caries under a designated fissure. Regardless, Penning found specificities comparable to previous studies. The sensitivities were found to be significantly lower than that of previous studies cited in the paper, yet all sensitivities were found to be no better than 62% (Penning, van Amerongen et al. 1992). Penning reports that one clinical study revealed a sensitivity of 82% with a specificity of 100%, however failed to mention how these were calculated . In 1993, Lussi compared different methods of diagnosis for non-cavitated fissured surfaces. In this in vitro study, 100 human teeth with no caries on smooth surfaces were selected (Lussi 1993). Of the original 100 teeth, 63 (52 molars, 11 premolars) were found to have macroscopically intact occlusal surfaces. Dentists were informed that all teeth came from teenagers with average caries experience. They were given 20 seconds

21

to examine each tooth, and to make a specific diagnosis for a designated spot on the occlusal surface. Each tooth was inspected using either: visual inspection; visual inspection with a magnifying glass; visual inspection combined with conventional bitewing radiography; visual inspection combined with light pressure probing; and conventional bitewing radiography. Each dentist was asked to diagnose each specified spot as either: having no caries; caries confined to enamel; or caries beyond the dentinoenamel junction (Lussi 1993). After all examinations were completed, the teeth were sectioned and examined histologically. Examination by dentists resulted in: 6 teeth with no caries; 19 teeth with caries confined to enamel; and 38 teeth with caries into dentin. Histologic examination revealed: 22 teeth with no caries; 13 teeth with caries confined to enamel; and 28 teeth with caries into dentin. Radiographic examination revealed: 21 teeth with no caries; 9 teeth with caries confined to enamel; and 33 teeth with caries into dentin (Lussi 1993). Statistical analysis revealed that visual inspection with or without probing had the lowest sensitivities with .12 and .14 respectively. Visual inspection with magnification improved sensitivity to .20, yet it was not a significant difference. Significantly higher values for sensitivity were found only when radiographs were involved, with bitewing radiography and visual inspection with bitewing radiography yielding sensitivities of .45 and .49 respectively (Lussi 1993). Conversely, examinations which included radiographs presented with the lowest specificities, although none of them significant. All specificities ranged from .83 to .93 with bitewing examination being the lowest and visual inspection and visual inspection with probing being the highest (Lussi 1993). Once again this study is consistent with the aforementioned studies, which all result in visual/tactile examinations having strong specificity with poor sensitivity. It can be concluded that visual examination may not reliably find caries, but will likely minimize over diagnosis. Lussi suggests that a more objective means of caries detection, e.g. caries

22

detection devices may be the most suitable tool in detecting occlusal caries with higher sensitivity. In 2004 Ismail did a comprehensive review of the literature. This was not a systematic review for all evidence published on visual and visuo-tactile methods of caries detection, rather his review was focused on the content validity of a sample of caries detection criteria reported in MEDLINE and the Cochrane Collaborations Oral Health Group (CC-OHG) (Ismail AI 2004). One hundred thirty six articles were selected from PUBMED and 35 articles from CC-OHG. One of the more important conclusions drawn from this paper is that there is a huge difference in philosophies between Europe and the United States. European researchers have been more progressive including early signs of dental caries in their caries detection criteria, where the USA have focused on measuring the cavitated stage of caries, when the explorer sticks with visual signs of caries demineralization (Ismail AI 2004). This review confirms the lack of consistency regarding explorer use, and again confirms that explorer use adds little caries detection while posing a possibility of detriment to enamel surfaces. The paper concluded that, this paper underscores the need to define a criteria system for visual and visuo-tactile detection of dental caries that has content validity based upon current scientific evidence and the consensus of experts in the fields of cariology and restorative sciences. (Ismail AI 2004)

Radiographic Detection

Radiographs are one of the most common methods used by providers to aid in diagnosis of dental caries. The most common radiograph used is the bitewing technique. Bitewing examinations are widely used to detect caries that may not be noticed during a visual clinical examination (Kidd EA, Pitts NB 1990). Bitewing radiographs are also used to determine the depth of caries involvement. Most often, bitewing examination is

23

affiliated with the diagnosis of interproximal caries. It is strongly recommended that these radiographs be used in the diagnosis of occlusal caries into dentin (Pitts NB 1991). Ricketts et. al. compared the diagnostic yield in caries diagnosis from traditional D speed and E speed films for occlusal and interproximal caries (Ricketts DN, Whaites EJ et al. 1997). In this in vitro study, 96 extracted molars and premolars (48 premolars and 48 molars) were collected with a range of carious appearances ranging from sound to frank cavitation on occlusal and interproximal surfaces. The teeth were examined and classified into one of the following categories: sound; white spot lesion; brown spot lesion; stained; undermining discoloration of dentin; cavitation less than 0.7mm; or cavitation greater than 0.7mm (Ricketts DN, Whaites EJ et al. 1997). The teeth were positioned in acrylic arch trays to simulate a normal intra-oral anatomical relationship. Four teeth (2 molars and 2 premolars) were placed in their respective anatomical order within the four quadrants of the mouth (16 teeth total) and mounted into an articulator. Six pairs of jaws were fabricated and used for radiographic examination (Ricketts DN, Whaites EJ et al. 1997). Bitewing radiographs were made using D speed and E speed films. Five examiners interpreted the radiographs. All radiographs were viewed using a view box under optimal settings. The films were examined in two separate sittings in an attempt to minimize evaluator fatigue. Each proximal and occlusal lesion was scored as: sound; radiolucency confined to outer half of enamel; radiolucency into inner half of enamel; radiolucency in outer half of dentin; radiolucency into inner half of dentin. This evaluation process was repeated at a later time to assess intra-rater reliability. After radiographic examination/interpretation, all teeth were serially sectioned and examined. Histologic findings were scored according to the following: sound; enamel caries in outer half of enamel; enamel caries into inner half of enamel; dentinal caries in outer half of dentin; dentinal caries into inner half of dentin (Ricketts DN, Whaites EJ et al. 1997). The largest recorded depth amongst the sections served as the gold standard

24

for validating radiographic examination. The sensitivity values at all diagnostic thresholds and all tooth surfaces were low. In general, the specificity values were high showing few false positives. However, specificity values for occlusal caries diagnosis in molars were lower than for other surfaces (Ricketts DN, Whaites EJ et al. 1997). The findings of low sensitivity are consistent with the works of others (Russell, Pitts 1993, Ricketts DN, Whaites EJ et al. 1997). Ricketts points out that this may reflect the difficulty of diagnosing demineralized tooth tissue when x-rays have to pass through intact buccal and lingual enamel, and that there is histological evidence to support that lesions are actually larger than they appear radiographically (Gwinnett AJ 1971). Ricketts also pointed out that in this study the sensitivity of occlusal caries diagnosis in premolars was so poor that radiographic diagnosis is practically useless. Wenzel et. al. studied the comparison of visual examination, conventional film, and digital radiographic enhancement on the assessment of occlusal caries depth, and in comparison to histologic appearance of the same lesion (Wenzel, Fejerskov et al. 1990). Forty seven extracted premolars and molars were selected for this study. The clinical appearance of these teeth ranged from appearing to be non-carious to having a large cavitation due to caries. Four observers examined these teeth visually and scored them according to the following rank scale: 0) no caries; 1) caries in enamel, not cavitated; 2) caries in enamel, not cavitated, presents with broad dark line in fissure; 3) small cavitation; 4) large cavitation; 5) very large cavitation, likely reaching the pulp (Wenzel, Fejerskov et al. 1990). Dental radiographs were made. The teeth were also digitized. The radiographs were examined and scored according to the following rank scale: 0) no caries; 1) caries in enamel; 2) caries reaching the dentino-enamel junction; 3) caries in outer half of dentin; 4) caries in inner half of dentin (Wenzel, Fejerskov et al. 1990). The depths of the lesions were also quantified on the digital images by counting the number of pixels in an occlusal-pulpal direction. The process was then repeated after a minimum time of one

25

month. The teeth were then hemi-sectioned, and the extent of the caries was scored according to the following rank scale: 0) no caries; 1) caries in enamel; 2) caries reaching the dentino-enamel junction; 3) caries in outer half of dentin; 4) caries in inner half of dentin. Forty five of the initial 47 teeth were used. The histologic findings revealed that 5 teeth were caries-free (score 0), 8 teeth had caries confined to enamel (score 1), 7 had caries reaching dentino-enamel junction (score 2), 7 had caries in the outer half of dentin (score 3) and 17 had caries in the inner half of dentin (score 4) (Wenzel, Fejerskov et al. 1990). This study found statistically significant correlations between clinical and histologic examinations, radiographic and histologic examinations, and digital radiographic and histologic examinations, with the latter having the strongest correlations. It is important to note that there was less agreement between traditional radiographs and histologic depth when compared to the correlation between clinical examination and histologic depth. It was largely due to the fact that some teeth were scored as caries free radiographically, when the lesion was actually deep into dentin (scores 3 or 4) (Wenzel, Fejerskov et al. 1990). It was found that the depth of the lesion measured through digital radiography was in good accordance with the lesion as it was found histologically. This reinforces Wenzel et. al.s idea that digital processing may be a good aid in accurately quantifying occlusal caries depth in a clinical setting. Wenzel and Fejerskov examined the accuracy of visual inspection, conventional radiography, and digital radiographic methods in the diagnosis of non-cavitated occlusal lesions (Wenzel, Fejerskov 1992). Additionally, the study investigated whether two or more of the previous methods provided an additive effect in the detection of occlusal caries . One hundred twenty-four fully erupted third molars were radiographed and extracted. This extracted sample was screened for the presence of cavitated lesions leaving 78 teeth which met the criteria for this study. All teeth were examined by visual inspection using air to dry the teeth, but without the use of a probe, and scored according

26

to the following scale: 0) no caries; 1) chalky/and occasionally stained fissure indicative of an early enamel lesion; 2) chalky and dark-stained fissure and a greyish shadow, indicative of a dental lesion, but with no evidence of cavitation along the fissure entrance; 3) same criteria as 2, but with small surface defects (Wenzel, Fejerskov 1992). The conventional film radiographs taken prior to extraction were examined using a light-box and viewer. The scoring criteria were as follows: 1) no caries or changes confined to enamel; 2) caries reaching dentin, but involving just the outer half; 3) deep dentinal caries, half-way or more to the pulp. The images were then digitally recorded and enhanced and scored using the same criteria that was used for scoring the conventional film radiographs. The teeth were then serially sectioned, with each section ranging between 500-600 !m in thickness. The sections were scored using the following criteria: 0) no caries; 1) enamel caries; 2) caries reaching dentin, but involving just the outer half; 3) deep dentinal caries, half-way or more to the pulp. The histologic examination revealed 4 teeth to be completely caries-free, 22 teeth with enamel caries, 24 teeth with shallow dentinal lesions, and 28 teeth with deep dentinal lesions. By comparison, visual inspection indicated that there were 33 teeth with dentinal lesions (5 of these were false-positive). Of the 28 teeth with deep dentinal lesions, visual inspection only made the same diagnosis for 15 of them. Conventional radiography (CR) indicated that 30 teeth had caries into dentin (5 of these were false-positive). CR only indicated deep caries in 8 of the 28 teeth with deep dentinal lesions, however 14 of them were indicated to have lesions into dentin. Digital enhancement indicated that 34 teeth were carious (6 of these were falsepositive). Fifteen of the 28 deep dentinal lesions were indicated as such through histological verification. When conventional radiographs were added to visual inspection results, the true positive detection rate increased by 11% with an increase in false positives to 7%. When

27

digital enhanced radiographs were added to visual inspection there was a gain of 33% in true positive detection with an increase in false positives of 11%. The results of this study indicated that visual inspection with digital radiographic examination may provide better accuracy in occlusal caries diagnosis than the other methods being tested(Wenzel, Fejerskov 1992). Russel and Pitts did a preliminary in-vitro study where they looked at the sensitivities, specificities, predictive value positives, and diagnostic accuracies of conventional bitewing radiography (D speed and E speed) and Radiovisiographs (Russell, Pitts 1993). One hundred twenty extracted posterior teeth were collected and mounted into silicone blocks. Four teeth were mounted per block (two molars and two premolars) in an attempt to mimic a posterior quadrant. Two blocks of four teeth were mounted in a relationship that mimiced an opposing upper and lower jaw, creating 15 artificial patients. Bitewing radiographs were made using: conventional D speed film; conventional E speed film; and Radiovisiographic images. For the conventional film, film settings were modified to optimize the results for the respective films (Russell, Pitts 1993). The radiographs were scored accordingly: R0) Sound tooth; R1) Outer half enamel lesion; R2) Inner half enamel lesion; R3) Outer half dentin lesion; R4) Inner half dentin lesion (Russell, Pitts 1993). The teeth were serially sectioned and examined independently. The histological scoring criteria is as follows: 0) no caries; 1) carious lesion in outer half of enamel; 2) carious lesion in inner half of enamel, but not in dentin; 3) carious lesion into outer half of dentin; 4) carious lesion into inner half of dentin (Russell, Pitts 1993). The results seem to indicate a slightly higher sensitivity with radiovisiography than with conventional radiography for the diagnosis of occlusal caries, however it was not found to be significant, nor were there any differences between either conventional radiograph or radiovisiography with regards to specificity, predictive value positive, or diagnostic accuracy. It is important to note that the sensitivity was found to be quite low

28

for all methods with regards to diagnosing occlusal lesions. The author notes that by using histological appearance to validate the accuracy of carious lesion detection, very early carious lesions are the likely cause of the low sensitivity (Russell, Pitts 1993). This article reinforces Wenzels notion that digital processing of radiographic images may lend to a more accurate estimation of occlusal caries depth than conventional radiography.

Caries Detection Devices

Fiber Optic Transillumination (FOTI)

As discussed earlier, the presence of lesions is influenced by changes in enamel structure. Enamel that has been disrupted due to the demineralization process will scatter photons of light differently than sound intact enamel. It is this scattering of light that creates the clinical presentation of white spots on the tooth surface. Fiber Optic Transillumination (FOTI) is used to enhance the clinical presentation of these white spots due to scattered light. By concentrating a high intensity light on the tooth surface, the light is permitted to shine through the tooth, highlighting changes in enamel and dentin. The devices strength is its ability to help discriminate between early enamel and early dentin lesions (Pretty 2006). It has been noted in some studies that FOTI diagnosis by eye can be subject to considerable intra- and inter-observer variation (Sidi AD, Naylor MN 1988, Verdonschot, Bronkhorst et al. 1992, Verdonschot EH, Wenzel A et al. 1993). To overcome these difficulties with FOTI, Digital Imaging Fiber-Optic Transillumination (DIFOTI) records images with a Charge-Coupled Device (CCD) imaging camera, instantaneously. This enables findings to be controlled and repeatable (Schneiderman, Elbaum et al. 1997).

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Cortes, et. al. compared the performance of visual examination using fiber optic transillumination to traditional visual examination, and bitewing radiographs to detect and estimate the depth of occlusal caries (Crtes, Ekstrand et al. 2000). In this study, 59 unrestored molars were selected. The occlusal anatomy of each tooth was diagramed, and a selected site was identified and marked on the drawing. Each examiner scored the occlusal surface with respect to the area marked in the drawing on a rank scale as follows: 0) no or slight change in enamel translucency after prolonged air drying (5 seconds); 1) opacity or discoloration hardly visible on the wet surface, but distinctly visible after air drying; 2) opacity of discoloration distinctly visible without air drying; 3) localized enamel breakdown on opaque or discolored enamel and/or greyish discoloration from underlying dentin; 4) cavitation in opaque or discolored enamel exposing dentin (Crtes, Ekstrand et al. 2000). After four hours the same teeth were examined using FOTI and scored according to the following ranked scale: 0) no shadow or stained area; 1) thin grey shadow appears when transilluminated; 2) wide grey shadow appears when transilluminated; 3) orange brown shadow appearing to be in dentin, 2mm or less in diameter; 4) orange brown shadow appearing in dentin greater than 2mm in diameter (Crtes, Ekstrand et al. 2000). Radiographs of the teeth were made and examined by one examiner, who was not involved in the visual scoring. The ranked scale used to score the teeth was: 0) no radiolucency; 1) radiolucency visible in enamel; 2) radiolucency visible in the dentin but restricted to the outer 1/3 of dentin; 3) radiolucency extending to the middle 1/3 of dentin; 4) radiolucency in the pulpal 1/3 of dentin. The teeth were then sectioned into three 250 !m sections. The most extensive changes were used to score according to the following ranked scale: 0) no demineralization; 1) outer ! enamel demineralized; 2) inner ! enamel demineralized; 3) outer 1/3 dentin demineralized; 4) middle 1/3 dentin demineralized; 5) inner 1/3 dentin demineralized (Crtes, Ekstrand et al. 2000).

30

Cortes et. al. examined the 59 teeth and found histologically that: 6 teeth were caries free, 10 had caries in outer half of enamel, 24 had caries in inner half of enamel, 13 had caries in outer third of dentin, 4 had caries in the middle third of dentin, and 2 had caries in the inner third of dentin. It was found that the highest correlation was found between visual detection and histological scores, followed by FOTI with histological, and radiographic detection and histological respectively. These differences were not statistically significant, however it was noted that the major significance was the poor ability to detect enamel lesions radiographically. All methods were quite good at detecting deeper lesions located into dentin, but had difficulties with determining depth of a lesion in enamel or outer third of dentin (Crtes, Ekstrand et al. 2000). The study confirmed that FOTI is as accurate as a detailed visual inspection, however it is important to note from the previously cited literature in this thesis, that visual inspection is not a great indicator for small lesions due to its poor sensitivity and inconsistent reproducibility. In another study, Cortes et. al. compared the combination of FOTI and traditional visual inspection to traditional visual inspection by itself, FOTI by itself, DIAGNOdent and Electrical Caries Monitor (ECM) (Crtes, Ellwood et al. 2003). This section will focus on the visual examinations, while the other caries detection devices will be addressed in subsequent sections. In this study, 152 sites were used from 111 extracted molars. The occlusal surfaces of the 111 teeth were photographed and the sites of interested were indicated on a drawing of the tooth. The visual assessments were scored according to the criteria seen in (Table 12). The teeth were then serial sectioned and evaluated by one evaluator. The caries involvement was scored on a seven point scale based on depth of carious lesion. The seven categories were: Sound; Outer ! of enamel; Inner ! of enamel; At the DentinoEnamel Junction (DEJ); Outer 1/3 of dentin; Middle 1/3 of dentin; Inner 1/3 of dentin. The histological assessment found that, of the 152 sites, 34 (22%) were sound; 18 (12%)

31

were in the outer half of enamel; 24 (16%) were in the inner half of enamel; 38 (25%) were at the DEJ; 15 (10%) were in the outer third of dentin; 12 (8%) were in the middle third of dentin; and 11 (7%) were in the inner third of dentin. Of the 34 sound sites, only 13 (38%) were scored sound using only the visual method and only 17 (50%) using the FOTI and combined FOTI and visual. The visual method identified 21 (55%) of dentin lesions correctly and the FOTI and combined FOTI and visual 25 (66%) and 26 (68%) respectively. The highest correlations with the histological scores were seen for the combined FOTI and visual and FOTI at (0.66) and (0.64) respectively (Crtes, Ellwood et al. 2003). FOTI, Visual and combined FOTI and visual showed high sensitivity and low specificity for enamel lesions and high specificity and low sensitivity for dentin lesions. It was concluded from this study that FOTI/visual combined method may be superior to visual assessment, and that the effectiveness of all methods of assessment were reduced when in the presence of stain and brown spot lesions (Crtes, Ellwood et al. 2003). Similar to Cortes et. al., Ashley et. al. did an in vitro study where they compared the Electronic Caries Monitor (ECM), FOTI and traditional and digital bitewing radiography to one another using histological findings to validate findings (Ashley PF, Blinkhorn AS et al. 1998). We will focus on the findings related to the methods previously covered and discuss ECM in a subsequent section. One hundred and three permanent teeth(68 premolars and 35 molars) were selected for this study. The visual assessment was performed after drying the tooth with compressed air and scoring according to the criteria developed by Downer, 1975. Similar to the visual assessment, examination was done using FOTI after drying the tooth with compressed air. Caries was evaluated and recorded according to the criteria adapted by Houwink et. al in 1970. Radiographs for each tooth were made using standard radiographic film and direct digital imaging. After completion of all examinations, each tooth was serially sectioned at approximately 0.4 mm intervals. All sections were examined under stereomicroscope

32

and classified using the criteria developed by Downer, 1975. The histological examination revealed 41 teeth had sound occlusal surfaces, 25 had evidence of occlusal caries extending into enamel only and 37 had occlusal caries extending into dentin (Ashley PF, Blinkhorn AS et al. 1998). The prevalence of disease in the extracted teeth was 60% with only two teeth having caries which extended beyond 1/3 the total depth of dentin. The results illustrated that visual diagnosis had one of the lowest specificities along with the ECM (0.73) but a slightly lower sensitivity (0.60) and lower positive (0.77) and negative predictive value (0.55) for enamel lesions. FOTI and both radiographic techniques both had higher specificities (0.80-0.88) than visual diagnosis and ECM, but lower positive (0.60-0.72) and negative predictive values (0.40-0.42), and much lower sensitivities (0.19-0.24 (Ashley PF, Blinkhorn AS et al. 1998)). For lesions into dentin the ECM provided the highest combination of sensitivity (0.78) and specificity (0.80) and the highest negative predictive value (0.87). Visual diagnosis had the highest positive predictive value (0.82) and specificity (0.97). The ECM had the lowest specificity (0.80) than the other systems (0.89-0.95), but the other systems had much lower sensitivities (0.14-0.24). FOTI had the lowest combination of sensitivity and specificity. Visual diagnosis was the least repeatable measurement with a low value of kappa (0.42). Overall, FOTI and radiographs performed poorly in this study. The sensitivities and specificities were significantly different from those for the ECM at both diagnostic levels (Ashley PF, Blinkhorn AS et al. 1998).

DIFOTI

Fifty extracted teeth including: 16 incisors, 8 canines, 12 premolars, and 14 molars were collected and mounted in modeling stone. Each tooth was inspected clinically by 2 experts using x4 magnification and an explorer, and histologic sections were used as the

33

gold standard. These teeth were all scored systematically using DIFOTI. Radiographs were made of all 50 teeth and examined as well. Five clinicians did the radiological examinations with four of them having training in DIFOTI. Each of the five clinicians used radiographic interpretation and DIFOTI techniques. These were scored and compared to the gold standard in order to determine sensitivity and specificity. For occlusal lesions this study found the sensitivity of DIFOTI to be over 3 times higher than radiographic examination with a specificity approximately 10% lower. It was concluded that DIFOTI had superior diagnostic capabilities when compared to radiographs, particularly in early, incipient lesions, however it has a greater tendency to over diagnose when no disease is in fact present.

Laser Fluorescence

Laser light is composed of electromagnetic waves with equal wavelengths and phases (Fejerskov 2009). Materials, including tooth structure, possess the characteristic of fluorescence. In fluorescence, emitted light has certain properties (wavelength) which increases as it is absorbed within the material. The larger wavelength is caused by loss of energy incurred during the absorption process. By using filtering techniques, this wavelength can be measured. The measurement recorded is proportional to the physical properties of the material. When the properties are known, for example sound enamel and dentin, it can be used as reference. Any change from this reference value can imply a deviation from normal tissue. Phenomena such as demineralization or bacterial presence can influence this change. Change in fluorescence radiance and lesion area can be followed in time to measure lesion development. The amount of fluorescence radiance loss is related to the mineral loss in the lesion. Therefore, change in fluorescence can be used as a diagnostic tool to identify the change in surface properties of tooth structure.

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Quantitative Laser Fluorescence (QLF)

De Josselin de Jong et. al. looked at the development of laser fluorescence methods in an attempt to assess initial enamel caries lesions in vivo. Young teenagers scheduled for orthodontic therapy were selected for this study. In each patient, two intact premolars were selected. In each case these teeth were previously treatment planned for extraction. Custom orthodontic brackets were fabricated and a plastic plate was fixed to the bracket at approximately 1mm distance from the tooth surface partially covering the buccal enamel surface. The space between the plastic and enamel enabled plaque stagnation area which was allowed to mature between four and six weeks (de Josselin de Jong, Sundstrm et al. 1995). Video recordings of laser fluorescence images of the buccal enamel surfaces were taken at the beginning of the experiment. The same recording was taken four to six weeks later upon the removal of the orthodontic brackets and the removal of plaque. These surfaces were then recorded again after another three and five weeks. It was concluded in this study that the method of using QLF to measure mineral changes in vivo was suitable and may be useful in clinical trials. It is important to note however, that this study was limited to smooth surface lesions, and may not be applicable to pit and fissured surfaces(de Josselin de Jong, Sundstrm et al. 1995). Ando et. al. compared QLF with dye-enhanced laser fluorescence (DELF), transverse microradiography (TMR), and confocal laser-scanning microscopy (CLSM) to detect and quantify mineral loss in artificial lesions in vitro (Ando, Hall et al. 1997). One hundred and forty four bovine enamel specimens were collected. A 3mm section was prepared and used for this study. Each specimen was covered in nail polish, leaving a .8mm x 2.0mm window in the center. The specimens were divided into 14 groups and placed in a demineralizing solution. Three groups containing 6 specimens were each placed in demineralizing solution for 2, 4 and 24 hours, respectively. Ten groups

35

containing 12 specimens were each placed in demineralizing solution for 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 hours, respectively. A further group of 6 specimens acted as a negative control and were not exposed to demineralizing solution. Upon completion of demineralization, the polish was removed and all specimens were analyzed with all of the aforementioned methods (Ando, Hall et al. 1997). All specimens were able to be analyzed using QLF and DELF, however only 130 of the specimens could be used to analyze using TMR and CLSM due to the inability to get thin enough sections. Because QLF is the emphasis of this section, we will only discuss the results pertaining to QLF. QLF appeared to be a useful tool for quantifying mineral loss. It was noted that this method, shows considerable scope as a clinical and laboratory tool for measuring mineral loss in carious lesions. It was also noted however, that QLF did not show the ability to record no mineral loss in specimens not exposed to demineralizing fluid therefore effecting its specificity (Ando, Hall et al. 1997). Similar to Andos study, Al-Khateeb, et. al. studied QLF in a more portable, clinical friendly light source in an attempt to validate the observed changes in fluorescence compared to data obtained from TMR. The methods for this experiment were done in both bovine and human enamel, however emphasis will be placed on the human enamel in this section. Thirty sound extracted premolars were gathered and cut into blocks. The blocks were covered in nail polish except for a 2mm x 3mm window. The blocks were submerged in alternating demineralizing and remineralizing solutions until artificial lesions were produced. Once the lesions were deemed adequate for the study, the samples exposure to demineralizing solution was dropped to two, 10 minute exposures daily, with the blocks in remineralization solution the remainder of the day. The blocks were scanned with the QLF during and after the lesion formation, and once every fourth day during the remineralization period for 60 days. At the end of the study period, all enamel blocks were sectioned and analyzed with TMR (al-Khateeb S, ten Cate JM et al. 1997).

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During the remineralization period, the fluorescence radiance of the enamel was partly regained in all enamel samples. A highly significant correlation was found between fluorescence and TMR. This solidifies the concept that the QLF readings could be used as a predictive value for caries depth but not necessarily a good gauge for change in mineral content. Despite the solid correlation, a very accurate calculation of mineral loss was not able to be calculated. This paper concluded that this new clinic friendly portable device was comparable with previously tested QLF protocols (al-Khateeb S, ten Cate JM et al. 1997) Ferreira Zandona et al. studied the effects of combining the ICDAS standardized visual system with QLF to determine the ability to monitor caries progression. Five hundred sixty nine children ranging from 5 to 13 years of age who presented with at least 1 permanent molar that had at least 1 unrestored surface were selected for this study. All children received a baseline DMFT, an ICDAS exam covering all surfaces off all teeth, and an exam of occlusal surfaces of molars with the QLF which also included buccal surfaces for mandibular teeth and lingual surfaces for maxillary teeth. ICDAS and QLF examinations were repeated 8 and 12 months later along with another DMFT at the 12 month interval (Ferreira Zandon, Santiago et al. 2010). Of the 569 children that participated at baseline, 460 children completed their 12 month exam. The average DMFT at baseline was 6.0 and was 6.4 at 12 months. The QLF examinations scored more early lesions than the ICDAS. The QLF also scored more lesions (almost 50% more) believed to be greater than half way through dentin than ICDAS. Both methods were able to follow the increase in average DMFT scores from baseline (Ferreira Zandon, Santiago et al. 2010). While it is important to note that the aim of the study was to look at the combined method of using visual criteria with QLF as an adjunct, the data supports that the QLF is more acute at finding subclinical changes that may only be detectable histologically. Given this evidence, it was discussed that the detection of early lesions is of little significance if the lesions are not in fact active and/or

37

not progressing. Finding these lesions without determining activity does not provide any meaningful information to a clinician, however it can be inferred that the ability to detect the lesion, and check the change in mineral content over time would be a useful tool in determining the proper treatment modality.

Light Induced Fluorescence (DIAGNOdent)

Lussi et. al. did an in vitro study where they compared the validity of the KaVo DIAGNOdent to the Electronic Caries Monitor (ECM). The aim of the study was to assess the validity while determining cut-off points of the new device for different stages of the caries process, and evaluating the reproducibility (Lussi, Imwinkelried et al. 1999). One hundred five teeth with intact occlusal surfaces were selected for this study. Occlusal surfaces were assessed using electrical conductivity measurements with the Electronic Caries Monitor (ECM) and with laser fluorescence (DIAGNOdent). Histologic examination was used as the gold standard to validate the reliability of both devices. Of the 105 teeth examined, histological examination revealed that: 21 teeth were caries free, 15 teeth had caries extending up to halfway through the enamel (D1), 31 teeth had caries in the inner half of enamel (D2), 28 teeth had caries in the outer half of dentin (D3), and 10 teeth had caries in the inner half of dentin (D4). The optimal cut-off limits for the laser device were as follows: 0-4 = no caries or caries to (D1); 4.01-10 = caries confined to (D2); 10.01-18 = caries limited to (D3); >18 = caries extending to (D4) (Lussi, Imwinkelried et al. 1999). Findings indicated that specificity for laser fluorescence values ranged between 72% (dried teeth, D2 level) and 87% (moist teeth, D3 level). For sensitivity, the respective values were 76% (moist teeth, D3 level) and 87% (moist teeth, D2 level). The ECM showed values between 64% (specificity, D2 level) and 92% (sensitivity, D3 level) (Lussi, Imwinkelried et al. 1999).

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In the discussion of this paper, it is identified that the higher specificity values for the laser fluorescence at the D2 level compared to ECM, combined with the identical sensitivity values at the same level of moist teeth is indicative of a more accurate diagnosis of initial carious lesions. Additionally, while the sensitivity values are somewhat lower for the DIAGNOdent compared to the ECM at the D3 level on moist teeth, the DIAGNOdents specificity is considerably higher. It was concluded that the high reproducibility of the DIAGNOdent combined with its high caries detection ability, laser fluorescence may prove to be a valuable tool for the longitudinal monitoring of caries and for assessing the outcome of preventive interventions (Lussi, Imwinkelried et al. 1999). Lussi et al. also did an in vivo study where they tested the DIAGNOdent in order to determine if the device should be recommended for use in the dental office. For this study, seven practicing dentists in Switzerland and Germany examined 240 patients with a mean age of approximately 20. A total of 332 occlusal surfaces were examined. Only molars (74%) and premolars (26%) with visibly intact occlusal surfaces were used for this study. All teeth were air dried and examined visually. Radiographs were used when available. Based on visual/radiographic examination, the dentist made a determination regarding the need for surgical intervention. Each tooth was also scanned using the DIAGNOdent. Upon surgical intervention, each tooth was determined to be free of caries using an explorer. The presence and extent of carious lesions were classified as follows: enamel caries (D1,D2); superficial dentinal caries (D3); deep dentinal caries (D4) (Lussi, Megert et al. 2001). The results determined that caries extended into enamel in 29 teeth, superficial dentinal caries in 146 teeth, and deep dentinal caries in 49 teeth. One hundred and eight teeth were considered to be caries free, exhibiting any signs of discoloration, opacities, staining, or any signs of caries clinically or radiographically (Lussi, Megert et al. 2001).

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Based on the results, the following readings on the DIAGNOdent were associated with the following states of caries: Values 0-13: no caries; values 14-20: enamel caries; values >20: dentinal caries (Lussi, Megert et al. 2001). The overall performances of all three diagnostic methods are summarized as follows: Visual inspection had a sensitivity of 31% (D3-level) and 62% (D2-level), whereas the DIAGNOdent device showed a sensitivity of >92%. Based on the results of this study the following guidelines for clinical use of the DIAGNOdent were recommended with the intent of minimizing the number of possible false positive readings: Values 0-13, no active care advised; Values 14-20, preventive care advised; Values 21-29, preventive or operative care advised depending on patients risk assessment; Values >30, operative care is advised (Lussi, Megert et al. 2001). Based on the findings in this study, the authors determined that the DIAGNOdent went seemed unsuitable for the detection of initial carious changes in enamel. It was also determined that the device was not able to distinguish between truly deep and superficial carious lesions. It was concluded, and subsequently confirmed with other literature which states that, visual inspection at occlusal sites provides insufficient sensitivity and is inferior to the sensitivity of the DIAGNOdent. The laser device can be a valuable adjunct tool for a second opinion in caries diagnosis. Shi et al. studied the reproducibility of the KaVo DIAGNOdent in occlusal caries detection in vitro. They used microradiography to validate their caries detection method and determined threshold values for enamel and dentinal lesions (Shi, Welander et al. 2000). Seventy-six extracted posterior teeth (48 molars and 28 premolars), which presented with intact occlusal surfaces, were selected for this study. Each tooth was numbered and measured using the DIAGNOdent. Three scans were taken when the tooth was wet, and three after the tooth had been wiped with a tissue and air dried. The maximum value while scanning was registered and the mean values of the three data

40

points under wet and dry conditions were calculated. This procedure was repeated after a two week interval. All occlusal surfaces were photographed (Shi, Welander et al. 2000). The teeth were radiographed and examined by 6 dentists. The observers were instructed to rate the radiographic examination and score based on the following criteria: 1 = definitely not caries; 2 = probably not caries; 3 = questionable; 4 = probably caries; 5 = definitely caries (Shi, Welander et al. 2000). The teeth were then cut into bucco-lingual sections of approximately 300 !m thick. The sections were cut perpendicular to the occlusal surfaces. The slices were microradiographed, and the films were examined under a stereomicroscope with a magnification of 16. The lesion depth was scored according to the following scale: 0 = sound; 1 = enamel caries limited to the outer half of the enamel; 2 = enamel caries in the inner half of the enamel; 3 = dentinal caries in the outer half of the dentin; 4 = dentinal caries in the inner half of the dentin (Shi, Welander et al. 2000). The study found the DIAGNOdent to be highly reproducible, but there were significant findings to support that the DIAGNOdent was influenced by humidity, presence of stain, calculus, etc. The principal limitation of the method was than an increased reading could reveal any change in physical properties of the tooth structure and not be limited to caries involvement. It was concluded that the DIAGNOdent system is superior to conventional radiographic methods, and the results are highly reproducible (Shi, Welander et al. 2000). As discussed earlier, Cortes, et al. compared combinations of FOTI and visual inspection to DIAGNOdent and ECM. When examining the rank correlation with histologic findings, the DIAGNOdent was found to have the lowest score of any other method, however its correlation was still considered statistically significant (p<0.001). In enamel lesions the DIAGNOdents sensitivity scored the lowest with a value of 0.73 compared to ECM which scored 0.80, and all visual examinations which were > 0.97. The specificity of the DIAGNOdent in enamel lesions was superior to all with a score of

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0.85. The next highest specificity was the ECM with a value of 0.71, and all visual inspections no greater than 0.50 (specificity of pure visual inspection was 0.38). In dentin lesions the DIAGNOdents sensitivity was superior to all other methods examined with a sensitivity of 0.84. The next highest value was a 0.68 which was observed with the ECM and a combination of FOTI with visual. As one might expect, DIAGNOdents specificity in dentin lesions was the lowest with a value of 0.67. All other methods had values > 0.90 When occlusal surfaces without the presence of stain and/or brown spot lesions were examined, there were improvements in the performance of the DIAGNOdent. In enamel lesions, sensitivity stayed virtually the same 0.72, the specificity improved to 0.91. In enamel lesions the sensitivity improved to 0.93 and the specificity improved to 0.72. The findings in this study support the notion that DIAGNOdent performance is reduced in the presence of stain and/or brown spot lesions. Devices not reliant on visual examination, may require a different set of cut-offs to compensate for stain and/or discoloration when using them (Crtes, Ellwood et al. 2003).

Electrical Conductance

Every material has its own electrical signature which means the properties of that material dictate the amount of electrical current which passes through them . The properties of a normal tooth are such that dentin is more conductive than enamel. It can be concluded that when affected by caries, tooth structure (enamel or dentin) has an increased porosity resulting in a higher fluid content. The presence of this fluid within the tissue improves electrical conductivity. This change in conductivity can be detected and measured. This quantifiable value can then be used to determine the presence of dental caries.

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Ricketts, et. al. did a study in 1995 where they looked at electrical resistance measurements both in vivo and in vitro on occlusal caries using the Vanguard Electronic Caries Detector and the Caries Meter L. Neither machine was commercially available as of the study, but it was tested due to the continued interest in electrical resistance measurements as a means to diagnose dental caries. At the time of this study, the ECM was in development and hit the commercial market shortly after. For this study, forty teeth treatment planned for extraction from 20 patients, were selected. All patients had pre-operative radiographs made of the teeth set for extraction. A diagram of the occlusal pit and fissure system was drawn for each tooth. A clinical examination was performed and all teeth were classified as one of the following: sound fissure; stained fissure; white spot lesion at entrance of fissure; brown spot lesion at entrance of fissure; undermining stain shining through intact enamel (Ricketts DN, Kidd EA et al. 1995). Since none of the radiographs showed any enamel caries, the teeth were classified as either radiographically sound or having dentin caries. Resistance measurements were taken at each selected site using the Vanguard Electronic Caries Detector. The teeth were then extracted. Resistance measurements were taken again at the same site using the same device. The Caries Meter L was also used to take resistance readings. The teeth were sectioned and investigated using macroradiographs. The teeth were classified as: sound, caries in outer third of enamel, caries in middle third of enamel, caries in inner third of enamel, caries in dentin (Ricketts DN, Kidd EA et al. 1995). The results found a statistically significant relationship between Vanguard readings in vivo and in vitro with a correlation of 0.84. Identical readings were identified 72% of the time, concluding in vitro findings were as reliable as the ones found in vivo. Visual examination revealed a sensitivity and specificity of 27% and 89% respectively when the diagnostic threshold was set at enamel caries. The sensitivity and specificity was 78% and 53% respectively with a threshold set at deep enamel lesions or

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dentin lesions. When only dentin lesions were classified as caries the sensitivity and specificity values were 3% and 97% respectively. The sensitivity and specificity values of radiographic examination were 6% and 100% respectively for enamel caries and 13% and 100% for dentin caries. The Vanguard examination revealed 81% and 78% sensitivity and specificity for enamel lesions and 97% and 56% respectively for dentin lesions. The Caries Meter L was reported to have the same order of magnitude for sensitivity and specificity as the Vanguard (Ricketts DN, Kidd EA et al. 1995). The study concluded that electrical resistance showed promise in the future of caries detection however the specificity was low. At the time it was predicted that this could be improved with further developments. It was also noted that findings of low electrical resistance is a good indicator of demineralization, however it by no means was a valid indicator of caries activity. Caries activity would still need to be identified in order to determine a method of treatment (Ricketts DN, Kidd EA et al. 1995).

Electronic Caries Monitor (ECM)

The ECM Device employs a single, fixed-frequency alternating current which attempts to measure the bulk resistance of tooth tissue (Longbottom C, Huysmans MC 2004). The ECM probe is directly applied to a site in question, which is measured over a five second measurement period. Compressed air is emitted from the tip of the probe. This sequence is known as a drying profile which aids in collecting information regarding the characteristics of the lesion. The ECM then records the level of electrical impedance as dictated by the degree of porosity in the tooth structure therefore generating a score which can be interpreted as caries progression. Ricketts et al. did a study in 1997 where they aimed to determine the accuracy and reproducibility of a modified technique using ECM for the diagnosis of occlusal surface

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caries of posterior teeth. Rather than relying on airflow to remove fluid from pits and fissures in an attempt to calculate the conductance of a given site on a tooth, this alternative technique involved covering the entire pit and fissure system with a jelly that acted as a contact medium allowing an individual to get a caries reading for the whole tooth rather than a site specific portion of it. A drop of contact medium (KY Jelly, Johnson & Johnson Ltd., Maidenhead, UK) was impregnated with a red dye and applied to the occlusal surfaces of 96 extracted teeth (48 premolar, 48 molar). The selection criteria for these teeth were that they presented with no existing restorations. The occlusal surfaces were coated with the contact medium and examined using the ECM. The scores were recorded and this process was repeated on one-third of the total sample (n = 32) at least one week after initial readings (Ricketts DN, Kidd EA et al. 1997). After all readings were taken, the teeth were serially sectioned into 4-6 sections and examined using X3 magnification. The histologic appearance of the occlusal surfaces was scored as: sound, caries confined to enamel, caries in to dentin. The greatest measurement observed was used as the histologic finding. The histologic findings revealed that 6 (13%) molar surfaces were sound, 12 (25%) had caries confined to enamel only, and 30 (63%) had caries extending into dentin. For the premolars, 15 (31%) surfaces were sound, 26 (54%) had enamel caries only, and 7 (15%) had caries extending into dentin (Ricketts DN, Kidd EA et al. 1997). The sensitivity and specificity for the overall readings of the entire sample at the D1 (enamel caries) threshold were 61% and 68% respectively. At the D3 (caries in dentin) threshold, the sensitivity and specificity values were both 76%. Findings indicated that electrical resistance measurements were better on molars than premolars at the D1 threshold with a sensitivity and specificity of 81% and 86% respectively. There was little difference found between molars and premolars at the D3 threshold (Ricketts DN, Kidd EA et al. 1997). Rickets et al. concluded from this study that this technique had potential to produce acceptable diagnostic accuracy and reproducibility.

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In 1998 Huysmans et al. aimed to determine in an in vitro study the reproducibility of the aforementioned modified technique using ECM where they coated the occlusal surface of posterior teeth with conducting gel. In this study, they also examined the correlation between ECM measurements and histological lesion depth, and whether there was a difference between premolars and molars based off of the idea that premolar teeth yield lower conductance readings than molars due to smaller size and subsequent electrode surface (Huysmans, Longbottom et al. 1998). Sixty-eight extracted teeth (34 molars and 34 premolars) with no restorations or frank cavitation on their occlusal surfaces were selected. The teeth were collected in sets of four teeth (2 molars, 2 premolars) and mounted into blocks to simulate normal anatomical position. Eight operators (3 experienced and 5 inexperienced) performed measurements on all teeth. Repeated measurements were made on a randomly selected subset of teeth (6 blocks; 24 teeth) on the same day. After all teeth were measured, the 68 teeth were added to teeth from two other studies bringing the total tooth count up to 325. All 325 teeth were serial sectioned and viewed under a stereomicroscope at 12-20X magnification. Caries observed microscopically were scored on the following scale from 0-4: 0 = sound, 1 = caries lesion in outer half of enamel, 2 = caries lesion in inner half of enamel, 3 = caries lesion in outer half of dentin, 4 = caries half in inner half of dentin. Histologically, 48 teeth (31 premolar, 17 molar) were sound, 60 teeth (51 premolar, 9 molar) had a score of 1, 82 teeth (43 premolar, 39 molar) had a score of 2, 107 teeth (21 premolar, 86 molar) had a score of 3, and 28 teeth (2 premolar, 26 molar) had a score of 4 (Huysmans, Longbottom et al. 1998). The mean intra-examiner reproducibility for the inexperienced operators was 0.81 and the mean for experienced operators was higher at 0.95, although not statistically significant, implying that experience with the device was not indicated to get reliable readings with this experimental method. The correlation coefficients were -0.78, -0.64, and -0.73 for all teeth, premolars, and molars respectively. These results demonstrate

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that the correlation using the combined sample demonstrated a moderate to good correlation between histological depth and electrical measurements (Huysmans, Longbottom et al. 1998). It was concluded that the ECM had a very good reproducibility, even in the hands of an inexperienced operator. The correlation between histologic lesion depth and ECM measurements was fair to good. It should be noted that there does appear to be differences in electrode area differences between premolars and molars resulting in the need to have different cut-offs based on the tooth being examined (Huysmans, Longbottom et al. 1998). Revisiting the study done by Ashley et al. who compared the ECM to other caries detection methods as discussed in previous sections, one hundred three permanent teeth with intact occlusal surfaces absent of any restorations were selected for this in vitro study (Ashley PF, Blinkhorn AS et al. 1998). The results in the histologic examination revealed 41 teeth with sound occlusal surfaces, 25 with evidence of caries extending into enamel and 37 with evidence of caries extending into dentin. In enamel lesions, the ECM provided the highest combination of sensitivity and specificity (0.65 and 0.73) respectively (despite having the lowest overall specificity). In dentin lesions it also provided the highest combination of sensitivity and specificity (0.78 and 0.80) respectively. The ECM was found to be the most repeatable measurement with the highest kappa value at (0.63) and weighted kappa of (0.68). Despite its repeatability, having the lowest overall specificity in both enamel and dentin lesions means it is the most likely to indicate surgical intervention on sound surfaces (Ashley PF, Blinkhorn AS et al. 1998). Revisiting Cortes et al. similar to the Diagnodent, the ECM was influenced by the presence of stain and brown spot lesions. It was concluded that specific cut-offs were required to use the respective devices. In the authors discussion it was mentioned that rigidly adhering to the cutoffs is not advisable when using ECM. The example given is

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that if >20 is the cutoff for dentin caries, one should be hesitant to commit to surgical intervention in the event the reading on the ECM was a 21. It was concluded that an obvious weakness of using ECM is the need to accommodate for tooth presentation by modifying the cutoff points on the device. Even when cutoffs are applied, they are still susceptible to subjective interpretation rendering the device unreliable as an objective measure of dental caries (Crtes, Ellwood et al. 2003).

CarieScan PRO

The CarieScan PRO is a handheld caries detection device which is applied to a tooth surface after it is isolated and properly dried. The probe tip of the device remains on the tooth surface for about 3-5 seconds. A score ranging from 0 100 will be generated. The scores are based on probability of caries with a score ranging from 0-50 resulting in a low probability; a score ranging from 51-90 resulting in a moderate probability; and a score ranging from 91-100 resulting in a high probability of the presence of caries in the location in contact with the device. Red, Yellow, and Green LED pyramids are illuminated on the device to correspond with the numerical score. As one would expect the green pyramid can be seen with scores from 0-50, the yellow with scores from 51-90, and red with scores from 91-100 (Figure 8). Like ECM, the CarieScan PRO measures resistance through tooth structure in an attempt to quantify levels of demineralization. As stated earlier, ECM relies on fixedfrequency alternating current to measure bulk resistance of the tooth, where the CarieScan PRO uses Electrical Impedance Spectroscopy (EIS) to determine the electrical properties of the tooth. The main advantage of EIS over a fixed-frequency measurement is that EIC can help to determine more accurately the various parameters associated with anatomical differences, and subsequent conductive properties, of different components of teeth (Longbottom C, Huysmans MC 2004). Put simply, by looking at electrical

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resistance in tooth structure coming from several waves of various frequencies of energy, one can record more information about the tooth than if one were to simply send a single frequency of energy through the same tooth structure. The manufacturer of this device claims that this new approach to caries detection is highly successful, resulting in a sensitivity of approximately 95% and a specificity of approximately 93%. Most of the literature available for this device was found as abstracts and table clinics. Due to its relatively short time on the market, there is little literature available to unequivocally support the manufacturers claims. Pitts et al. submitted an abstract in Caries Research [41(4):321-322 (2007)] where the aim of the study was to assess the probability of the correct determination of transitions, or stasis, of repeated lesion scores derived from a pre-production ac-Impedance Spectroscopy (ac-IS) device in vitro. One hundred two teeth were studied by 2 dentists who made 3 assessments each. A reference standard was carried out using dental examiners assessing tooth surfaces using micro CT in addition to an optimal clinical visual technique. The study found the probabilities of assessing the correct determination of transitions from repeated measurements to be 94% for occlusal surfaces, 94% for free smooth surfaces and 94% for interproximal surfaces. It was concluded that this device could be useful in monitoring serial changes in the caries status of teeth due to its high reproducibility in transitions. Longbottom et al. submitted an abstract in Caries Research [41, p297 (2007)] where they compared the diagnostic performance of several diagnostic techniques including: ICDAS, Bite Wing radiography, Laser Fluorescence using the Diagnodent pen, and acimpedance spectroscopy. These methods were validated by comparing against a reference assessment using a combination of clinical examination with micro-CT. Using a Success Ratio at the D1 threshold which was calculated using the: number of correct measurements divided by the total number of measurements, the ac-impedance technique had the highest success ratio percentage on occlusal surfaces at 93% followed by ICDAS

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at 80%, laser fluorescence at 48%, and bite-wing radiography at 26%. The sensitivity and specificity was also calculated and scored as follows: ac-impedance 97/30, ICDAS 98/50, bite-wing radiography 80/100, and laser fluorescence 36/75. This study concluded that at the D1 threshold, ac-impedance has a superior detection performance compared to other techniques. Pitts et al. submitted another abstract in Caries Research [42(3), p211 (2008)] where 137 extracted teeth were examined using the CarieScan PRO and compared to a reference evaluation which consisted of micro CT images and visual examination. Teeth were scored as: Green (G) = sound; Yellow (Y) = caries present where preventive care is advised; and Red (R) = caries present where operative care is advised. For the occlusal surfaces, the sensitivity was calculated as a result of R versus (G + Y) when compared to a measurement, which would present with the actual presence of disease, and totaled 92.5%. The specificity was calculated as a result of G versus (Y + R) when compared to a measurement, which would present with the actual presence of disease and totaled 92.5% and the accuracy for Y alone was 79.4%. Hall et al. submitted an abstract in Caries Research [41(4):296 (2007)] where they determined the intra- and inter-examiner repeatability of an ac-impedance spectroscopy device in vivo. Four dentists examined five surfaces on two adjacent teeth in 19 different patients. To determine inter-examiner repeatability, two additional groups of four dentists examined five surfaces on two adjacent teeth in each of a different group of 19 patients using an ac-IS device. Surfaces were scored as either: (G), (Y), or (R). The Intra-examiner agreement was 73.98%, and the Inter-examiner agreement was 70.94%. It was concluded that this device demonstrates substantial agreement for both intra- and inter-examiner repeatability.

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CHAPTER III MATERIAL AND METHODS

The aim of this chapter is to provide a detailed description of the design of this study, along with all materials used to ensure its completion. All indices and statistical analysis used have been specifically defined to ensure absolute reproducibility.

Tooth Selection

One hundred twenty five extracted human teeth from the University of Iowa College of Dentistry were randomly selected for this in vitro study. The inclusion-criteria for this study were as follows: Permanent posterior teeth, which presented with intact occlusal surfaces, no occlusal restorations, and no obvious cavitation in pits & fissures or on the proximal or buccal or lingual surfaces. Occlusal staining was permitted in this study provided it did not accompany frank cavitation and/or exposed dentin. These teeth were cleaned using a straight edge razor and a tooth brush. After cleaning, all teeth were stored in a 1% Thymol solution to prevent any bacterial growth prior to and during the study. Any teeth with obvious cavitation to their occlusal surfaces were removed from the collection. Each tooth was labeled so that it could be identified at a later time.

Digital Macro Photographs of Occlusal Surfaces

A Nikon D80 Digital Camera equipped with a Nikon, AF-s Micro Nikkor 105 mm 1:2.8 G VR lens and a Sigma EM-140 DG ring flash were used to photograph all occlusal surfaces. The camera was mounted onto a tripod (tiltall #4602 professional), and tilted downward so that the camera faced the floor. A weighing boat was rested on

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the adjustment knob of the tripod and stabilized with rope wax. The weighing boat was used as a stage to place teeth for photographing. The camera body was placed in manual mode. The flash was also placed in manual mode at ! for 1 and 2. The lens was adjusted to an f-stop of 51, a shutter speed of 125 and an ISO of 100. Rope wax was manipulated into a cube and rested on the weighing boat in frame to be photographed. Each tooth was removed from its storage container and rinsed of storage medium prior to being placed in the rope wax and photographed immediately (Figure 1). The tooth was than dried with compressed air for 5 seconds and photographed a second time. The 5 second air drying was done in accordance with ICDAS protocol for examining teeth as alluded to in the previous section (www.icdas.org). The wet and dry photographs of each tooth were cropped uniformly and placed side-by-side into a PowerPoint presentation (Microsoft PowerPoint 2010). Each occlusal lesion of interest was identified with a circle in the PowerPoint presentation. Each tooth was assigned a number (Figure 2).

Scoring Occlusal Lesions

The presentation was sent to three ICDAS trained and calibrated examiners (JK, WS, and GD) in order to score the lesions. They scored independently and recorded the ICDAS code for the lesion of interest. To evaluate intra- and inter-examiner reliability a second scoring was necessary. In order to assess the intra- and inter-examiner reliability without introducing bias into the study, the teeth in the PowerPoint presentation were assigned a number and were then re-sequenced using a random sequence generator (www.random.org) (Figure 3). The PowerPoint presentation was re-sequenced according to the random sequence generator and sent back to the three examiners one week after the initial scoring. The

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independent scoring and coding were once again returned and were recorded. These recorded scores were used to determine intra-rater, and inter-rater reliability. When universal agreement was not achieved by all three examiners, they viewed and discussed the lesions together and came to a consensus. The consensus score was used in association with the CarieScan PRO, and with histologic examination. This method will be discussed in a later section. The ICDAS criteria for pit & fissured surfaces can be seen as follows:

ICDAS II Criteria Code

Code 0: Sound Tooth Surface: No evidence of caries after prolonged air drying (5s). Code 1: First visual change in enamel: opacity or discoloration (white or brown) is visible at the entrance to the pit or fissure after prolonged air drying, which is not or hardly seen on a wet surface. Code 2: Distinct visual change in enamel: opacity or discoloration distinctly visible at the entrance to the pit and fissure when wet, lesion must still be visible when dry. Code 3: Localized enamel breakdown due to caries with no visible dentin or underlying shadow: opacity or discoloration wider than the natural fissure/fossa when wet and after prolonged air drying. Code 4: Underlying dark shadow from dentin with/without localized enamel breakdown (Figure 4) .

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Caries Analysis Using CarieScan PRO

Charging the CarieScan PRO

Before using the CarieScan PRO, the battery was charged for at least 4 hours per manufacturers instructions. The main plug from the power supply unit was plugged into an ac socket. The dc jack from the power supply unit was inserted into the power socket at the rear of the charging cradle. The CarieScan PRO, was placed into the cradle. The blue LED indicator on the CarieScan PRO was illuminated when charging was initiated (Figure 5). System Testing

A system test was performed at the beginning of each day, and each time a sensor collar to the device was replaced during testing. This was done at a regular interval during the experiment and will be discussed in further detail later. When applicable, the existing collar, housing the sensor, was removed. The cable test adaptor (used for system tests) to the CarieScan PRO was inserted on the neck of the device, locking it into place. The device was turned on. The lip hook connector cable was connected into the side of the device and into the pin of the test adaptor. The test was initiated, resulting in an ok every time it was used. The cable test adaptor was removed and the collar was replaced with a new sensor (Figures 6&7).

Scoring Teeth with the CarieScan PRO

Ninety five teeth in total were scanned for this experiment. Each tooth was removed from its storage container. The root of each tooth was placed into contact with the lip hook of the CarieScan Pro device. The hook and root were wrapped in a piece of

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gauze soaked in artificial saliva to replicate the oral environment. Following the manufacturers instructions, each tooth was air dried using a 3-in-1 syringe for 5 seconds. The CarieScan PRO sensor was placed on the tooth site to be measured. The site of interest to be measured was confirmed using the photographed/circled images from the PowerPoint that was previously used for scoring the site of interest using ICDAS. The measurement scan began as soon as the device came in contact with the tooth surface. To complete its scan, the sensor is held in place for approximately 4 seconds. The resulting value was recorded by the principle investigator. The site of interest was measured five times. All readings were between 0 and 100. The mean of the 5 scores was recorded. The mean score was categorized into one of the devices seven pre-set color codes. The range of codes for each (color) category is listed below (Figure 8): Green: 0 Yellow 1: 1 20 Yellow 2: 21 30 Yellow 3: 31 50 Yellow 4: 51 90 Yellow 5: 91 99 Red: 100 Sensors were replaced after scanning 20 teeth, to ensure excessive wear to the sensor did not affect the devices ability to properly scan the tooth surfaces of interest. By systematically replacing the sensor, we were able to control the amount of wear on the sensor with respect to the tooth surface of interest. That is to say, the sensor was used for the fifth time when scanning tooth A5 the first measurement and the second measurement, eliminating the chance that the condition of the sensor affected the outcome of scans on a given surface. Upon, replacing the devices sensor, the system test of the device was performed to make sure there were no malfunctions. Upon scoring of all teeth, the procedure was repeated by the principle investigator (JC) one week later.

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The mean scores were compared from the two time intervals to evaluate the intra-rater reliability of the device (Figure 9). All measurements were made at approximate room temperature (70-73 degrees F) with a humidity range of 20%-22%

Tooth Sectioning

Tooth Preparation for Sectioning

The area of interest was located on the occlusal surface as it appeared in the PowerPoint document to ensure that the proper area would be examined histologically. The teeth were oriented so that the buccal and lingual margins of each tooth ran parallel to the top and bottom of the computer. A 21mm 020 Flexofile (Dentsply) was placed across the area of interest perpendicular to the buccal and lingual marginal ridges. The location of the file was marked with red nail polish (Sinful Colors, Dream On 113) on the buccal marginal ridge and green nail polish (Sinful Colors, San Francisco 283) on the lingual marginal ridge. This was done to guide sectioning at the area of interest (Figure 10). After the polish was dry, the occlusal surface of each tooth was acid etched with 37% phosphoric acid for 30 seconds (Figures 11&12). The etch was washed thoroughly with water for 20-30 seconds and air dried completely. Optibond FL adhesive was placed with a brush on the occlusal surfaces and polymerized for 20 seconds using a Tungsten/Halogen curing light (Kerr Demetron 100-120V~/2.0 AMP 50/60 HZ) (Figure 13). A flowable composite was placed along the occlusal surface, making sure to keep the nail polish dots exposed. The composite was polymerized for 40 seconds (Figure 14).

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Mounting Teeth For Sectioning

Each tooth was placed, roots first into a cylinder designed for the SilverstoneTaylor Series 1000 Deluxe Hard Tissue Mircotome at the University of Iowa College of Dentistry (Figure 15). If the roots did not fit in the lumen of the cylinder, modifications to the root were made with a diamond bur in a high-speed dental handpiece. The tooth was fixated with red sticky wax and was tested to ensure that it was immobile. Each tooth was placed so that the CEJ of each tooth was exposed. When inadequate root was available for good retention, grooves were placed in the root with a high-speed handpiece. The exposure of the complete anatomical crown was imperative to ensure that all tooth sections went to the pulp chamber (Figure 16).

Sectioning

The cylinder was locked into the microtome. The motor speed was increased to 20,000 revolutions per minute. The feed rate of the microtome was set at 5 (out of 10). Four cuts were made in the tooth, across the buccal and lingual marginal ridges in an apical direction to the depth of the CEJ, resulting in 3 sections (Figure 17). Each cut was made at 600 micron intervals, creating sections of approximately 400 microns in thickness. The microtome was set up so that the section created from the second and third cut, the middle section, would contain the red and green dot placed on the buccal and lingual marginal ridges. A section was also created both mesial and buccal to this section as a result of the 1st and 2nd, and the 3rd and 4th cut respectively. Due to the thickness of the blades, each section thickness was approximately 400 microns (Figure 18). All cuts were made through the pulp chamber so that the full extent of dentin could be examined histologically (Figure 19).

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Removing Sections

The three sections from every tooth were carefully removed using a straight edged razor blade and an Interproximal Carver. The razor blade was initially placed in the spaces between sections, while gently applying apical pressure. As the spaces widened slightly the interproximal carver was placed in the same manner, until the sections separated (Figure 20). The sections were placed in the same direction with the mesial

side facing up and in the same order as they sat in the crown prior to sectioning. All three sections were placed on a glass slide with the first section removed from the tooth placed on the far right and the third section on the far left (Figure 21). The sections were examined under Polarized Light Microscopy.

Polarized Light Microscopy

All sections were examined under an Olympus BH-2 Polarized Light Microscope equipped with a trinocular eyepiece and a rotating stage. Attached to the microscope was an Olympus DP25 Camera. All sections were photographed and analyzed in Stream Basic 1.4 software. The sections were examined under approximately 10x magnification. The photograph dimensions of each section were 2560 x 1920, with an exposure time of 1/39 seconds at a subject distance of 5mm (Figure 22). Each section was labeled according to the order they were photographed. The order they were photographed was in accordance to their orientation with respect to the other sections. Therefore the section on the right was the section on the right on the slide. By doing this, it was easy to determine the change in a lesion from a mesial/distal direction if desired (Figure 23).

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Histologic Examination

All three sections per tooth were looked at when examining each tooth. In some instances only two sections were recovered, and in two other instances there were four sections obtained from the tooth. The four samples were a result of poor initial placement of the microtome, making it necessary to cut an additional section to ensure the lesion was recorded with sections mesial and distal to the sectioned indicated by nail polish. It was quite difficult to get the section marked by nail polish in the middle section consistently. Often the nail polish area fell across multiple sections and as a result, all sections were used to determine the depth of caries involvement. The principle examiner developed a PowerPoint presentation to be used as a tutorial on how to histologically score the sections. Two examiners (JC and JK), one of which was the principle examiner, trained on the tutorial and discussed scoring criteria prior to scoring histological sections. The sections were independently examined and scored by the two examiners. The teeth were scored according to a ranked scale used by Crtes et. al and is described below (Crtes, Ekstrand et al. 2000) (Figure 24). One week later, the teeth were again reexamined and scored by the same examiners. The scores were examined for intra-rater and inter-rater reliability. Where universal agreement was not achieved by both examiners, they discussed the lesions together until they came to a consensus.

Ranked Scale

0: No demineralization 1: Outer enamel demineralization 2: Inner enamel demineralization

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3: Outer 1/3 demineralization of dentin 4: Middle 1/3 demineralization of dentin 5: Inner 1/3 demineralization of dentin

Statistical Analysis

All tests employed a 0.05 level of statistical significance. SAS for Windows (v9.3, SAS Institute Inc., Cary, NC, USA) was used for the data analysis.

Intra- and Inter-Rater Reliability for Visual Inspection

The weighed kappa statistic was used to evaluate intra- and inter-observer agreement (comparison of ICDAS scores given at two different time points by the same rater and by different raters respectively). The nonparametric Wilcoxon signed-rank test was also used to detect the difference between the two measurements which were either made by the same rater or by the two raters. The closer the kappa value is to one, the greater the agreement between the two assessments. The following is an approximate guide for interpreting level of an agreement that corresponds to kappa coefficient: (Shrout PE and Fleiss JL, 1979): i) ii) iii) iv) v) vi) vii) 0 = No agreement 0 0.20 = poor agreement 0.21 0.40 = Fair agreement 0.41 0.60 = Moderate agreement 0.61 0.80 = Substantial agreement 0.81 0.99 = Strong agreement 1.00 = Perfect agreement

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Intra-Rater Reliability of Examination with the CarieScan PRO

The concordance correlation coefficient was computed as a measure of agreement between the two means, taken from the five measurements, made by the same observer (JC) at two different times. A paired-samples t-test was used to determine whether a significant difference existed between two duplicated measurements made on the same tooth by the single observer.

Intra- and Inter-Rater Reliability of Histologic Examination

The weighed kappa statistic was used to evaluate intra- and inter-observer agreement (comparison of histologic scores given at two different time points by the same rater or by the two raters respectively). The nonparametric Wilcoxon signed-rank test was also used to detect the difference between the two measurements which were either made by the same rater or by the two raters.

Association Between Visual Inspection and Histologic Findings

Descriptive statistics were conducted. Chi-squared test and Fishers exact test (i.e. when the sample size is relatively small and the expected frequency in any of the cells is less than five) were used to examine the statistical significance of an association between groupings of ICDAS codes (codes 0-2 and 3-4 as well as codes 0-3 and four), and histologic scores (histologic codes 0-3 and 4-5) (Table 14). The significance between the two different groupings of ICDAS codes is to try and address the criteria for surgical intervention in the United States and Europe. ICDAS code threes, are more likely indicated for surgical intervention in the United States than in Europe.

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Association Between CarieScan PRO and Histologic Findings

Descriptive statistics were conducted. Chi-squared and Fishers exact test (i.e. when the sample size is relatively small and the expected frequency in any of the cells is less than five) were used to examine the statistical significance of an association between groupings of mean CarieScan scores (scores 0-50 and 51-100 as well as scores 0-30 and 31-100) taken from the second measurement and a consensus of histologic scores (scores 0-3 and 4-5). The significance between the two different groupings of CarieScan scores is to try and address the criteria for surgical intervention in the United States. CarieScan scores greater than 30 are more likely to present with the possibility of significant carious change beneath the enamel surface according to the manufacturer, and likely indicated for surgical intervention. Scores greater than 50 are more likely to present with the probability of significant carious change beneath the enamel surface according to the manufacturer, and likely indicated for surgical intervention (Table 15).

Association Between Visual Inspection Scores and CarieScan PRO

Descriptive statistics were conducted. Chi-squared and Fishers exact test (i.e. when the sample size is relatively small and the expected frequency in any of the cells is less than five) were used to examine the statistical significance of an association between groups of ICDAS codes (codes 0-2 and 3-4 as well as codes 0-3 and code four) and mean CarieScan scores (scores 0-50 and 51-100) taken at the second measurement (Table 16).

Sensitivity and Specificity for ICDAS

The ICDAS and histologic data were placed into a 2x2 table, where histologic scores are used as a surrogate of truth. This table can be seen here (Table 1).

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Table 1. ICDAS vs Histo I

ICDAS vs Histo I ICDAS 0-2 ICDAS 3-4 Histologic 0-3 Histologic 4-5

The idea for creating these ICDAS and histologic groupings, is that histologic caries of a score greater than 4 (Histologic 4-5) is considered likely to require surgical intervention. By the same logic, a histologic score of three and below (Histologic 0-3) is considered not requiring surgical intervention. With respect to ICDAS scoring, ICDAS scores 0-2 are considered not requiring surgical intervention and therefore scores 3-4 may require surgical intervention. To interpret the accuracy of the ICDAS, the four interpretations of results are shown as follows (Table 2).

Table 2. ICDAS vs Histo I Interpreted

ICDAS vs Histo I Interpreted ICDAS 0-2 ICDAS 3-4 Histologic 0-3 True Negative False Positive Histologic 4-5 False Negative True Positive

The Sensitivity for ICDAS would be: True Positive/(True Positive + False Negative) and The Specificity for ICDAS would be: True Negative/(True Negative + False Positive). The ICDAS groups were changed to: ICDAS 0-3 and ICDAS 4 just as they were for the association calculations between CarieScan scores and ICDAS codes. The same data used in the previous table was also placed into the following (Table 3):

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Table 3. ICDAS vs Histo II

ICDAS vs Histo II ICDAS 0-3 ICDAS 4 Histologic 0-3 Histologic 4-5

Again, the idea for this table is the same as ICDAS vs Histo I table only the cutoff for surgical intervention, based on the ICDAS scoring, is 4.

Sensitivity and Specificity for the CarieScan PRO

The ICDAS and histologic data were placed into a 2x2 table which resembled the following (Table 4):

Table 4. CarieScan vs Histo I

CarieScan vs Histo I CarieScan 0-50 CarieScan 51-100 Histologic 0-3 Histologic 4-5

The idea for this table is that Histologic caries of a score greater than 4 (Histologic 4-5) is considered to require surgical intervention. By the same logic Histologic score of three and below (Histologic 0-3) is considered not requiring surgical intervention. With respect to CarieScan scoring, a score of 0-50 are considered initial lesions and therefore not requiring surgical intervention and therefore scores 51-100 are considered significant

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caries and may require intervention. To interpret the accuracy of the CarieScan, the four interpretations of results are shown as follows (Table 5):

Table 5. C.S. vs Histo I Interpreted

C.S. vs Histo I Interpreted CarieScan 0-50 CarieScan 51-100 Histologic 0-3 True Negative False Positive Histologic 4-5 False Negative True Positive

The Sensitivity for CarieScan would be: True Positive/ (True Positive + False Negative); The Specificity for CarieScan would be: True Negative/ (True Negative + False Positive).

The CarieScan data was also categorized as (Table 6):

Table 6. CarieScan vs Histo II

CarieScan vs Histo II CarieScan 0-30 CarieScan 31-100 Histologic 0-3 Histologic 4-5

The idea for this table is the same as the ICDAS vs Histo I table only the cutoff for surgical intervention based on the CarieScan scoring is greater than 30 which is defined as the possibility of significant carious change beneath the enamel surface according to the manufacturer.

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Pilot Study

Several pilot studies were performed in an attempt to improve proposed methodology prior to the main study. Thirteen different test configurations were used in conjunction with the microtome to best find an optimal protocol in tooth sectioning. Among these configurations were: placement of resin on the occlusal surface; reduction of cusps; and various section thicknesses. Modifications resulting from these pilot studies helped establish the testing methodology and measurement described in the materials and methods section of this study.

Hypotheses 1. There is no statistically significant correlation between the scores recorded using the CarieScan PRO and the scores recorded using the modified Cortes scale in histologic examination. 2. There is no statistically significant association between the scores recorded in ICDAS inspection and the scores recorded using the modified Cortes scale in histologic examination. 3. There is no statistically significant association between scores recorded in ICDAS inspection and the scores recorded using the CarieScan PRO.

Operational Definitions Alternating Current (AC): The movement of electricity in which the electric charge periodically reverses direction AC Electrical Impedance: A measure of opposition to Alternating Current Dental Caries: Dental caries, also known as tooth decay or a cavity, is a disease where bacterial processes damage hard tooth structure. This disease progression may exist as superficially as the outer enamel layers.

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LED: Light-emitting diode (LED) is a semiconductor light source. LEDs are used as indicator lamps in many devices, and are increasingly used for lighting Sensitivity: The probability that the test says a person has the disease when in fact they do have the disease. Specificity: The probability that the test says a person does not have the disease when in fact they are disease free.

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Figure 1

Tooth Mounted for Photographs

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Figure 2

Microsoft PowerPoint image of photographed teeth. Wet image on the left. Dry image on the right. Red circle denotes the area of interest

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Figure 3

Photograph of the Excel spreadsheet used to randomize the photographed lesions to prevent any bias during second evaluation

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Figure 4

ICDAS Codes 0 - 4

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Figure 5

CarieScan PRO fully charged in cradle per manufacturer's specifications

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Figure 6

Sensor collar in place calibrating the CarieScan PRO per manufacturer's specifications

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Figure 7

Proper placement of the CarieScan PRO sensor

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Figure 8 CarieScan color pyramid and corresponding numbers

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Figure 9

With the lip hook in contact with the tooth and wrapped in a gauze saturated in artificial saliva, the CarieScan PRO sensor is placed on the site of interest and a score is recorded

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Figure 10 Placing buccal (red) and lingual (green) marks to designate location of the lesion prior to covering occlusal surface with resin

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Figure 11 Etching the occlusal surface prior to placing resin

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Figure 12 Frosted appearance in the enamel after acid etching

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Figure 13 Placing an adhesive prior to resin placement on the occlusal surface

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Figure 14 Placing composite resin on the occlusal surface

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Figure 15 Series 1000 Deluxe Hard Tissue Microtome

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Figure 16 Mounting ring ready to be placed in microtome for sectioning. The tooth was mounted high enough in the wax to expose the CEJ

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Figure 17 One of four cuts in the tooth to get three sections

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Figure 18 Serial sections in a buccal/lingual direction through the site of interest which was identified by the paint

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Figure 19 Sections cut into the pulp chamber so that the extent of caries into dentin can be properly assessed

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Figure 20 Separating the second of three sections using the Interproximal Carver (IPC)

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Figure 21 Serial sections prepared for histologic exam with Polarized Light Microscopy (PLM). Sections placed on the slide in the order they were separated. The red paint can be seen on the buccal surfaces of the middle and left sections

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Figure 22 Looking at sections using an Olympus BH-2 Polarized Light Microscope

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Figure 23 Image taken from PowerPoint document used to score histologic lesions. The images from left to right correspond with the sections on the slide as seen in Figure 21

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Figure 24 Examples of histologic scoring using the Crtes ranked scale

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CHAPTER IV RESULTS Evaluations of Intra- and Inter-Observer Reliability for Measurements In this section, observers are identified by their initials: JK, GD, WS, and JC. Intra-Observer Reliability for ICDAS Scores For JK, the kappa coefficient of 0.69 (95% confidence interval (CI): 0.58-0.80) indicated substantial agreement between the two ICDAS classifications. The data showed that 71.6% of JKs ratings stayed the same. Additionally, the Wilcoxon signed-rank test indicated that there was a significant difference between the two measurements (p=0.0354). This difference indicates a difference in median rating scores between the two measurements. For GD, the kappa coefficient of 0.43 (95% confidence interval (CI): 0.30-0.55) indicated moderate agreement between the two ICDAS classifications. The data showed that 50.5% of GDs ratings stayed the same. Additionally, the Wilcoxon signed-rank test indicated that there was a significant difference between the two measurements (p=0.0001). This difference indicates a difference in median rating scores between the two measurements. For WS, the kappa coefficient of 0.57 (95% confidence interval (CI): 0.45-0.68) indicated moderate agreement between the two ICDAS classifications. The data showed that 55.8% of WSs ratings stayed the same. Additionally, the Wilcoxon signed-rank test indicated that there was a significant difference in median rating scores between the two measurements (p=0.0006).

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Inter-Observer Reliability for ICDAS Scores For JK vs. GD, the kappa coefficient of 0.69 (95% confidence interval (CI): 0.580.80) indicated substantial agreement between the two examiners second ICDAS classifications. The data showed that 71.6% of ratings were the same between JK and GD. Moreover, the Wilcoxon signed-rank test indicated that there was no significant difference between two ICDAS classifications made by JK and GD (p=0.3042). For JK vs. WS, the kappa coefficient of 0.63 (95% confidence interval (CI): 0.510.74) indicated substantial agreement between the two examiners second ICDAS classifications. The data showed that 61.1% of ratings were the same between JK and WS. Moreover, the Wilcoxon signed-rank test indicated that there was no significant difference between the two ICDAS classifications made by JK and WS (p=0.3145). For GD vs. WS, the kappa coefficient of 0.52 (95% confidence interval (CI): 0.41-0.64) indicated moderate agreement between the two examiners second ICDAS classifications. The data showed that 52.6% of ratings were the same between GD and WS. Moreover, the Wilcoxon signed-rank test indicated that there was no significant difference between the two ICDAS classifications made by GD and WS (p=0.8733).

Inra-Observer Reliability for CarieScan PRO Scores Since two means were being compared with each other, Concordance correlation coefficient was computed to assess intra-observer (JC) agreement between the two CarieScan PRO measurements made one week apart on the same sample. The concordance correlation coefficient of 0.90 (95% confidence interval: 0.85-0.93) indicated a strong agreement between the two CarieScan PRO measurements made by the single observer (JC).

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Additional analysis was conducted to determine if there was a significant difference between first and second measurements using a paired-samples t-test. The data revealed that there was no statistically significant difference between the two measurements (p=0.3719). Descriptive statistics of mean differences between the first and second measurements are summarized below (Table 13).

Intra-Observer Reliability for Histologic Scores For JC, the kappa coefficient of 0.88 (95% confidence interval (CI): 0.82-0.95) indicated strong agreement between the two histologic scorings. The data showed that 87.1% of JCs scorings stayed the same. Moreover, the Wilcoxon signed-rank test indicated that there was no significant difference between the two scorings (p=0.6133). For JK, the kappa coefficient of 0.71 (95% confidence interval (CI): 0.60-0.83) indicated substantial agreement between the two histologic scorings. The data showed that 74.2% of JKs ratings stayed the same. Moreover, the Wilcoxon signed-rank test indicated that there was a significant difference between the two measurements (p=0.0319). Inter-Observer Reliability for Histologic Scores The kappa coefficient of 0.72 (95% confidence interval (CI): 0.62-0.82) indicated substantial agreement between the two histologic scorings made by observers JK and JC. The data showed that 72% of ratings were the same between JK and JC. Moreover, the Wilcoxon signed-rank test indicated that there was no significant difference between the two measurements made by JK and JC (p=0.2082).

94 Evaluations of the Associations Between Histologic Consensus, ICDAS Consensus, and CarieScan PRO Mean Association of Histologic Consensus with ICDAS Consensus There was a significant difference in the six histologic consensus categories (histologic 0-5) between the two ICDAS categories (ICDAS codes 0-2 and 3-4) (p=0.0137; Fishers exact test). This analysis indicated that teeth that were in the ICDAS code 0-2 category, were most likely to have a histologic score of 3 (46.6%). The same histologic score of 3 was only found in the ICDAS 3-4 category 35% of the time. Teeth that were in the ICDAS code 3-4 category, were most likely to have a histologic score of 5 (40.0%). The same histologic score of 5 was only found in the ICDAS 0-2 category 15.1% of the time. The detailed frequency distributions are shown in Table14. There was a significant difference in two histologic consensus categories (histologic 0-3 and histologic 4-5) between the two ICDAS categories (ICDAS codes 0-2 and 3-4) (p=0.0012; chi-square test). This analysis indicated that teeth that were in the ICDAS 3-4 category were more likely to be in the histologic 4-5 category (65.0%) than teeth in the ICDAS 0-2 category (26.0%). The detailed frequency distributions are shown in (Table 14). There was no significant difference in the two histologic consensus categories (histologic 0-3 and histologic 4-5) between the two ICDAS categories (ICDAS codes 0-3 and code 4) (p=0.3352; Fishers exact test) (Table 14).

Association of Histologic Consensus with CarieScan PRO Mean There was a significant difference in the six histologic consensus categories between the two CarieScan mean categories (C.S. 0-50 and CS 51-100) (p<0.0001; Fishers exact test). This analysis indicated that teeth that were in the CarieScan (0-50) category were most likely to have a histologic score of 3 (53.5%).The same histologic

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score of 3 was only found in the CarieScan (51-100) category 28.6% of the time. Teeth that were in the CarieScan (51-100) category, were most likely to have a histologic score of 5 (40.0%) of the time. The same histologic score of 5 was only found in the CarieScan (0-50) category 8.6% of the time. The detailed frequency distributions are shown in (Table 15). There was a significant difference in two histologic consensus categories (histologic 0-3 and histologic 4-5) between the two CarieScan mean categories (C.S. 0-50 and C.S. 51-100) (p<0.0001; chi-square test). This analysis indicated that teeth that were in the C.S. 51-100 category were more likely to be in the histologic 4-5 category (62.9%) than teeth in the C.S. 0-50 category (17.2%). The detailed frequency distributions are shown in (Table 15). There was a significant difference in two histologic consensus categories (histologic 0-3 and histologic 4-5) between the two CarieScan mean categories (C.S. 0-30 and C.S. 31-100) (p=0.0013; Fishers exact test). This analysis indicated that teeth that were in the C.S. 31-100 category were more likely to be in the histologic 4-5 category (45.9%) than teeth in the C.S. 0-30 category (12.5%). The detailed frequency distributions are shown in (Table 15).

Association of CarieScan PRO Mean with ICDAS Consensus

There was a significant difference in the five ICDAS categories between the two CarieScan mean categories (C.S. 0-50 and C.S. 51-100) (p=0.0223; Fishers exact test). This analysis indicated that teeth that were in the CarieScan mean category (0-50) were most likely to have an ICDAS score less than 2 (18.6% for ICDAS 0 and 30.5% for ICDAS 1). The same ICDAS scores less than 2 (codes 0-1) were only found in the CarieScan mean category (51-100), 5.6% and 13.9% for ICDAS codes 0 and 1 respectively. On the other hand, teeth that were in the CarieScan Mean (51-100)

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category were more likely to be in ICDAS 2-3 categories (47.2% for ICDAS 2 and 27.8% for ICDAS 3). The same ICDAS scores 2-3 were only found in the CarieScan mean category (0-50), 37.3 and 8.5% respectively. The detailed frequency distributions are shown in (Table 16). There was a significant difference in two ICDAS categories (0-2 and 3-4) between the two CarieScan mean categories (C.S. 0-50 and C.S. 51-100) (p=0.0218; chisquare test). This analysis indicated that teeth that were in C.S. (51-100) catergory were more likely to be in the ICDAS 3-4 category (33.3%) than teeth in the C.S. (0-50) category (13.6%) The detailed frequency distributions are shown in (Tables 16). There was a significant difference in the five ICDAS categories between the two CarieScan mean categories (C.S. 0-30 and C.S. 31-100) (p=0.0084; Fishers exact test). This analysis indicated that teeth that were in the CarieScan mean category (0-30) were most likely to have an ICDAS score less than 2 (18.2% for ICDAS 0 and 42.4% for ICDAS 1). The same ICDAS scores less than 2 (codes 0-1) were only found in the CarieScan mean category (31-100), 11.3% and 14.5% for ICDAS codes 0 and 1 respectively. On the other hand, teeth that were in the CarieScan Mean (31-100) category were more likely to be in ICDAS 2-3 categories (51.6% for ICDAS 2 and 17.7% for ICDAS 3). The same ICDAS scores 2-3 were only found in the CarieScan mean category (0-30), 21.2 and 12.1% respectively. The detailed frequency distributions are shown in (Table 17). There was no significant difference in two ICDAS categories (0-2 and 3-4) between the two CarieScan mean categories (C.S. 0-30 and C.S. 31-100) (p=0.6166; chisquare test). Nor were there any significant differences in two ICDAS categories (0-3 and 4) between the two CarieScan mean categories (C.S 0-30 and C.S. 31-100) (p=0.9999; Fishers exact test) The detailed frequency distributions are shown in (Table 17).

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Sensitivity and Specificity Sensitivity and Specificity of ICDAS

In order to test sensitivity and specificity of a given system, one must be able to compare findings from that system to actuality. In this study, histologic findings were considered the truth, and histologic scores of 0-3 were considered to not require surgical/operative intervention. Histologic scores of 4-5 were considered to require surgical/operative intervention. ICDAS and CarieScan measurements were compared to these histologic findings and were validated according to them. From the 93 teeth scored to calculate sensitivity and specificity, the breakdown was as follows (Table 7):

Table 7. ICDAS vs Histo I

ICDAS vs Histo I ICDAS 0-2 ICDAS 3-4

Histologic 0-3 54 7

Histologic 4-5 20 12

With respect to the need for surgical intervention, visual detection lesions with ICDAS codes 0-2 would not require surgical/operative intervention. Those with an ICDAS code of 3-4 would require surgical/operative intervention. Fifty-four teeth (58.1%) were designated as a True Negative; 7 teeth (7.5%) were designated as a False Positive; 20 teeth (21.5%) were designated a False Negative; and 12 teeth (12.9%) were designated a True Positive. The Sensitivity for ICDAS was calculated at .375 or 37.5 %, and the Specificity for ICDAS was calculated at .885 or 88.5% The data was also placed in the following table with a different cutoff (Table 8):

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Table 8. ICDAS vs Histo II

ICDAS vs Histo II ICDAS 0-3 ICDAS 4

Histologic 0-3 59 2

Histologic 4-5 29 3

With respect to the need for surgical intervention, visual detection lesions with ICDAS codes 0-3 would not require surgical/operative intervention. Those with an ICDAS code of 4 would require a surgical/operative intervention. Fifty-nine teeth (63.4%) were designated as a True Negative; 2 teeth (2.2%) were designated as a False Positive; 29 teeth (31.2%) were designated a False Negative; and 3 teeth (3.2%) were designated a True Positive. The Sensitivity for ICDAS was calculated at .093 or 9.3 %, and the Specificity for ICDAS was calculated at .967 or 96.7%. Sensitivity ans Specificity of CarieScan PRO From the 93 teeth scored to calculate sensitivity and specificity, the breakdown was as follows (Table 9):

Table 9. CarieScan vs Histo I

CarieScan vs Histo I CarieScan 0-50 CarieScan 51-100

Histologic 0-3 48 13

Histologic 4-5 10 22

With respect to the need for surgical intervention, CarieScan PRO readings of 050 would not require operative/surgical intervention. Those with readings 51-100 would require operative/surgical intervention. As stated earlier, scores greater than 50 (51-100) are considered to have a, probability of significant carious change beneath the enamel

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surface according to the manufacturer. Forty-eight teeth (51.6%) were designated as a True Negative; 13 teeth (14.0%) were designated as a False Positive; 10 teeth (10.8%) were designated a False Negative; and 22 teeth (23.7%)% were designated a True Positive. The Sensitivity for the CarieScan PRO was calculated at .688 or 68.8%, and the Specificity for CarieScan PRO was calculated at .787 or 78.7%. The data was also placed in the following table with a different cutoff (Table 10):

Table 10. CarieScan vs Histo II

CarieScan vs Histo II CarieScan 0-30 CarieScan 31-100

Histologic 0-3 28 33

Histologic 4-5 4 28

With respect to the need for surgical intervention, CarieScan PRO readings of 030 would not require surgical/operative intervention. While ScarieScan PRO readings of 31-100 would require surgical/operative intervention. As stated earlier, scores greater than 30 (31-100) are considered to have the possibility of significant carious change beneath the enamel surface. Twenty-eight teeth (30.1%) were designated as a True Negative; 33 teeth (35.5%) were designated as a False Positive; 4 teeth (4.3%) were designated a False Negative; and 28 teeth (30.1%)% were designated a True Positive. The Sensitivity for the CarieScan PRO was calculated at .875 or 87.5%, and the Specificity for CarieScan PRO was calculated at .459 or 45.9%.

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Table 11

Nyvad et als description of diagnostic criteria from a 1999 paper in Caries Research

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Table 12 Criteria used for the visual, FOTI and radiographic examinations in Crtes et als 2000 paper in Caries Research

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Table 13 Descriptive statistics of mean differences between the first and second measurements with the CarieScan PRO

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Table 14 Associations of ICDAS levels with histologic consensus categories (N=95)

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Table 15 Associations of CarieScan levels with histologic consensus categories (N=95)

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Table 16 Associations of CarieScan levels (0-50, 51-100) with ICDAS levels (N=95)

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Table 17 Associations of CarieScan levels (0-30, 31-100) with ICDAS levels (N=95)

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CHAPTER V DISCUSSION

Statistical evaluation of the data rejects the first null hypothesis regarding the lack of association between CarieScan PRO scores and histologic categories. There was statistically significant evidence to support that when a tooth presented with a CarieScan score of 50 or under, the caries process extended no deeper than the outer 1/3 of dentin 83% of the time. When the tooth presented with a score greater than 50, the caries process was in the inner two-thirds of the dentin 63% of the time. When the CarieScan score cutoff was at 30 or under, lesions within the outer one-third of dentin could be seen 88% of the time; and when the score was greater than 30, the caries process was seen in the inner one-third of dentin 46% of the time. The change in CarieScan PRO score cutoff was to address the probability of caries activity per the manufacturers specifications. According to the CarieScan PRO pyramid defining the significance of CarieScan values (Figure 8), the cutoff separating the probability of initial lesions and significant carious lesions is at 30. As a result, scores greater than 30 are more likely to present with significant carious change according to the manufacturer. Even though they were found to be significant, the outcome of the data may present more ideally with larger sample sizes, particularly lesions which present as histologic 0s, and 1s. With a total of 4 Histologic 0s and 1s, greater numbers would have allowed for more powerful statistical analysis. There appears to be no literature studying the association between CarieScan readings and Histological scoring to compare these findings to. Statistical evaluation of the data both accepts and rejects the second null hypothesis regarding the association between ICDAS and histologic scores. There was statistically significant evidence to support that when a lesion presented with an ICDAS

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code of 0-2, the caries process extended no deeper than the outer 1/3 of dentin 74% of the time; and when a lesion presented with an ICDAS code of 3-4, the caries process could be found in the inner two-thirds of dentin 65% of the time. That is to say, smaller ICDAS codes (0-2) often yield shallower caries lesions, and the larger codes (3-4) yield deeper caries lesions. This also relates to ICDAS discriptors of the numerical code. For example an ICDAS code 2 is described as distinct visual change in enamel while an ICDAS code 3 is described as localized enamel breakdown due to caries with no visible dentin. There have been several studies which have affirmed the correlation between disease progression and ICDAS score as discussed in the literature review (Braga, Mendes et al. 2009, Nyvad, Machiulskiene et al. 1999). Conversely, when teeth presented with ICDAS codes of 0-3, caries presented no deeper than the outer 1/3 of dentin 67% of the time. This was not found to be statistically significant. The lack of sample sizes, particularly with teeth that presented as histologic 0s and 1s prevented us from examining the relationship between the individual five ICDAS codes with the five histological scores. There were also a small number of ICDAS code 4s gathered in this study. A larger sample size may have permitted this study to look at ICDAS code 3s on their own. The statistical significance found in the data tables (Table 14) may suggest that ICDAS code 3s may be more like code 4s than like code 2s. Having the ability to look at code 3s as a separate group, and compare ICDAS code3s to code 4s with respect to their association with histologic scores may be something worth exploring in future studies. The lack of statistical significance could have been influenced by the number of ICDAS 4 lesions examined. There were only 5 code 4s in total. While, we were able to use Fishers exact test to perform statistical analysis on the small sample size, its entirely possible that a greater sample of code 4s would increase statistical power and resulted in different findings.

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The reason for creating the different cutoffs in ICDAS scores has to do with the decision to treat surgically. In the United States, providers are more likely to treat lesions that present with any signs of loss of structure within the pits and fissures, potentially allowing for an explorer to stick. In Europe, providers are more likely to observe or provide non-surgical interventions to the same surface presentation (Ismail AI 2004). Based on this philosophical difference, an ICDAS lesion of 3 or greater would be possible grounds for surgical intervention in the United States, where a lesion of 4 or greater would be possible grounds for surgical intervention in Europe. It is possible that the reliability of the data collected could have an effect on the outcome. Statistical analysis concluded that there was substantial agreement between the two evaluators evaluating and scoring the histologic samples. Individually, the two evaluators had substantial agreement or better regarding their intra-examiner reliability. This success could be attributed to a tutorial on how to score histolologically, which was created by one observer (JC) and the amount of time the evaluators spent reviewing it. There was moderate to substantial agreement between the three evaluators in assigning ICDAS codes to the area of tooth that was of interest. Individually, the three evaluators had moderate agreement or better regarding their intra-examiner reliability. JK verbally discussed differences in examiner coding with GD, and WS via skype and telephone conversations. JK also reviewed the lesions and discussed in person with WS prior to the second evaluation. These additional discussions may have contributed to examiners re-calibrating themselves, and therefore resulting in different coding of the second evaluation. This would affect the intra-examiner reliability. The three evaluators, while formally calibrated on extracted teeth and live patients, were asked to evaluate photographs of occlusal lesions separately. Because the three evaluators live in three different cities, correspondence was done electronically. Evaluations were inadvertently performed under different circumstances; there was no control for: how long each examiner took to evaluate each of the 95 teeth; monitor size, type, or quality; whether or

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not they enhanced or enlarged photographs during evaluation, lighting conditions when evaluating lesions, etc. Additionally, despite being calibrated, the system used in this study is much more intricate than other systems that may only require that an examiner determine whether caries are initial, moderate, or severe like the ADA Caries Classification System (ADA CCS) (www.ada.org). The examiners were asked, in the ICDAS scale, to distinguish between lesions that would all be classified as initial or moderate in the ADA CCS scale for example. As a result, the three examiners have a much smaller margin of error in order to achieve high kappa values for reliability. These different conditions may explain the differences in inter-examiner reliability. They may have also affected the outcome of scores and subsequently had an effect on statistical analysis. Statistical evaluation of the data accepts and rejects the third null hypothesis regarding the association between ICDAS and CarieScan PRO scores. There was statistical significance to support that when CarieScan readings were 50 or below, the teeth presented with ICDAS codes 0-2 lesions 86% of the time. However, when the ICDAS cutoffs were changed (ICDAS 0-3 and 4), there were no significant findings. For CarieScan readings of 30 or below, there was statistical significance to support that ICDAS code 1 lesions were present 42% of the time. For CarieScan readings greater than 30, ICDAS code 2 lesions were present nearly 52 percent of the time. There wasnt any statistically significance evidence to support any association between CarieScan groups (0-30 and 31-100) with either ICDAS groupings (code 0-2 and 3-4 or code 0-3 and 4). The data from both Tables 16 & 17 suggest that there might have been significant differences in CarieScan groups (51-100) and (31-100) for ICDAS codes 2-3, which appear to be found at nearly 70%. As stated earlier, the low number of ICDAS code 4s may play a factor in the data collected. Its possible that there is simply poor association between these groups when the CarieScan cutoff of 0-30 and 31-100 is applied, but one would expect with good

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associations between ICDAS and histologic findings, and CarieScan PRO scores and histologic findings, that there would be a natural association between ICDAS and CarieScan PRO findings. Perhaps this statistic was adversely affected by small sample size of teeth with ICDAS codes 3-4. The CarieScan PRO has a 93% sensitivity for occlusal lesions according to the manufacturer. Even with a cutoff of 30, where anything greater was considered, possibility of significant carious change beneath the enamel surface according to the manufacturer, the data resulted in a sensitivity of 87.5%. The lower cutoff naturally yielded a higher sensitivity as the number of false positives (low CarieScan scores with high histologic scores) is minimized. With the cutoff at 50, where the manufacturer defines readings greater than 50 as probability of significant carious change beneath the enamel surface, the sensitivity was calculated at 68.8%. The CarieScan PRO has a 93% specificity for occlusal lesions according to the manufacturer. With a more optimal cutoff of 50, the results indicate a specificity of 78.7%. When using the cutoff at 30, the specificity drops to 45.9%. There are multiple factors that may account for the differences in the findings between this thesis and manufacturer claims (Huysmans MC, Longbottom C et al. 2000). The method of gathering data was different between the two studies, with the manufacturers reference study using 4 examiners to gather data, where only one was used for this study. One examiner may have been more reliable however, if the accuracy of these measurements was off, it could affect the outcome of the data therefore affecting the sensitivity and specificity. The occlusal surfaces in the reference study were collected while the tooth was in an environment with approximately 80% humidity and a temperature approximately that of body temperature. In this study, the humidity never exceeded 22% and the temperature at time of measure was never greater than 73 degrees Fahrenheit. Huysmans et al. identified temperature as a factor that affects electrical measurement (Huysmans MC, Longbottom C et al. 2000). Longbottom, et al. addressed

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these findings stating that although it was demonstrated that temperature affects electrical values obtained, the fact that this effect was linear simplifies in vitro to in vivo extrapolation of absolute values of parameters (Longbottom C, Huysmans MC 2004). While the two studies being compared are both in vitro, it must be considered as a variable possibly affecting outcomes between the two studies. Histological validation was done with PLM for this study compared to micro CT for the reference study. The method of statistical analyses may perhaps have the largest impact on the results between the two studies. The reference study used a probability map method to calculate its sensitivities and specificities. The histological cutoffs used to validate the CarieScan measurements are unknown as the only literature available is an abstract which does not present that information. As could be seen from the results of these studies, changes in cutoffs can affect the sensitivity and specificity results. Limited subjects with small histologic scores were present in this study forcing some histologic caries categories to be grouped. Larger sample size and further statistical analysis of ungrouped categories may have yielded data more closely resembling the findings from the reference study. This study was performed in the Midwestern portion of the United States, an area with optimal water fluoridation. The reference study was performed in Europe where the water fluoridation may not be as optimal. It is possible that the subject teeth and lesions present differently in different geographic regions. One can argue that there are less hidden caries in Europe due to the lack of fluoride. Teeth exposed to the caries process are more likely to lose enamel surface integrity in the absence of Fluoride resulting in cavitation. These teeth would be excluded in this study, leaving more teeth absent of disease or with more advanced disease easier to diagnose. There were a number of limitations in this study. Being an in vitro study, it is impossible to replicate in vivo conditions. In addition to not replicating real life situations with respect to temperature and humidity, experimenting on non-vital extracted

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teeth simply cannot produce the same resistance that one might encounter while taking ac impedance scans on living tissue. Because this study was dependent on the findings in extracted teeth, the available sample to choose from was limited. Many of the teeth that were selected ended up being histologic code 3s. The majority of these teeth were extracted third molars and pre-molars. In most instances, teeth are treatment planned for extraction as a result of non-restorability or inadequate space for proper function. Those that were non-restorable generally did not meet inclusion criteria and were therefore not available for this study. It can be inferred that the teeth that did meet criteria were either: impacted and not exposed to the oral environment and therefore not at risk of any demineralization; or exposed long enough to present with initial enamel breakdown but not long enough for activity to undermine the dentin. Based on the former, one would have anticipated more histological code 0s and 1. Working outside of synthesized oral environment may have been a limitation to this study aside from the fact that a pseudo-oral environment was not created. In absence of the high humidity, study teeth may have dehydrated more than what would occur in the mouth. This dehydration may have effected CarieScan scoring and the ability to assess its effectiveness. Due to financial constraints of this study, the protocol called for the re-use of CarieScan sensors. The method accounted for the wear when comparing teeth to themselves, however this could not account for the effect of the wear on the diagnostic ability for the device. Ideally, one sensor for every one to three teeth might have been a better method and changed the results of this study. Sectioning the teeth was also a challenge in this study. It was very difficult to preserve occlusal enamel using the available equipment at the University of Iowa College of Dentistry. The placement of composite resin on the occlusal surface proved to be incredibly helpful in preserving the pit and fissured, superficial enamel layers for study, however it came at a cost. Sectioning through the resin seemed to shorten the longevity

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of the microtome blades. These blades, appeared to bend, dull, and ultimately break much faster when cutting through resin and tooth structure than through just tooth structure. The destruction of these blades lead to the loss of 2 prepared samples and damage to several others. Frequent replacement of the blades would have greatly minimized this complication, however due to financial constraints, this was not possible. Additionally, due to the difficulty in sectioning enamel to assess occlusal lesions, the sections cut were quite thick compared to ideal diagnostic thickness using polarized light microscopy. The sample thickness for this study was 400 microns. At the College of Dentistry, the desired thickness for optimal PLM images is around 100 microns. During a pilot study, lesions were examined from sections of 400 microns in thickness, photographed, and then polished to 100 microns in thickness. The pilot study concluded that, while the 100 micron images were more clear and easier to look at, it did not affect the diagnostic quality for our study. As a result, the samples were left at a thickness of 400 microns. The results found from histologic examination may have been influenced by the primary examiner of this study. In an attempt to calibrate the two examiners to get an accurate and consistent yield in histologic findings, the primary examiner created a tutorial document, listing guidelines to follow when scoring the sections. The primary investigator had strong agreement between his first and second scorings, most notable because it was his criteria he used to score the sections. Other than the second examiner, the primary examiner was not calibrated with anyone else. Despite their precision in scoring, its possible that their accuracy was not very good. This would affect the outcome of the study. Despite the limitations in this study, there appears to be statistically significant evidence to suggest that there is an association between the findings in CarieScan PRO scores and the histological finding of caries. In general, low CarieScan scores seem to suggest the caries process is either confined to enamel, or superficial layers of dentin.

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These surfaces are often considered to not require surgical intervention in the absence of cavitation. Conversely, high CarieScan scores seem to suggest the caries process has progressed into the inner 2/3 of dentin. These surfaces are often considered requiring surgical intervention. Future studies might include another in vitro study with larger sample size. This would enable one to look at any correlation between CarieScan PRO readings and histologic findings, and not just associations as the result of using categorical data. Statistical significant data that could pinpoint histologic depth with a specific CarieScan reading or range would be very beneficial from both clinical and research standpoints. An in vivo study would also be beneficial in linking this studys findings to patient care. Confirmation of associations, even if not to manufacturers claims, would validate some practical application for the CarieScan Pro. Along with the CarieScan and Histologic data, the ICDAS data could also be used for follow up studies. Another follow up study would be to evaluate any histologic associations with specific ICDAS and CarieScan findings. One could assess the reliability of a hypothetical CarieScan reading in conjunction with an ICDAS score and its association with histologic findings and measure how that compares to the data found in this study. With discussion regarding the fluoridation standards in Europe and in the United States, comparison of ICDAS to histologic associations from the United States and Europe would help validate whether the ICDAS data (European literature) translates in a different geographic region. Based on the findings in this study, the CarieScan PRO would not be recommended as a primary method of diagnosing and assessing occlusal caries. Despite the association between CarieScan scores greater than 30 and histologic presence of caries into the inner two-thirds of enamel, this data is not strong enough to support manufacturers claims. Furthermore, the sensitivity and specificity findings in this study would indicate that CarieScan PRO readings would over-diagnose carious lesions,

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resulting in the overtreatment of teeth due to unnecessary surgical intervention. Further testing and larger sample sizes are clearly indicated in future studies before this device should be endorsed for anything more than an adjunct tool to visual inspection in the detection of occlusal caries.

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