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15 Endodontic Microbiology and Treatment of Infections

The basic science most closely associated with the practice of endodontics is microbiology. Most diseases of the dental pulp and the periradicular tissues are associated with microorganisms. Microbial invasion prompts the host to respond with a combination of nonspecific inflammatory processes and specific immunologic responses. Endodontic treatment, whether surgical or no surgical, essentially is debridement to disrupt and remove the microbial ecosystem associated with the disease process. Clinicians must understand the close relationship between the presence of microorganisms and endodontic disease processes to develop an effective rationale for treatment. Molecular methods now are used to study endodontic infections. These methods are much more sensitive and precise for detecting and identifying the organisms associated with endodontic infections. This chapter focuses on the role of microorganisms in the pathogenesis of endodontic infections and on effective clinical treatment of such infections. The theory of focal infection also is discussed; because of misinformation that has arisen on this subject, some are recommending extraction of teeth, rather than effective root canal treatment that preserves the natural dentition. ASSOCIATION OF MICROBES WITH PULPAL AND PERIRADICULAR DISEASES Colonization is simply the establishment of microbes in a host when biochemical and physical conditions are adequate for growth. Given the proper conditions, members of the normal oral flora may become opportunistic pathogens. Opportunistic pathogens cause disease if they gain access to normally sterile areas of the body, such as the dental pulp or periradicular tissues. The degree of pathogenicity caused by microbes is referred to as virulence. The pathogenic response includes damage caused by the host in response to the microbes. The host's response includes both nonspecific inflammatory and specific immunologic reactions.

Pathways of Pulpal Infection


Dental caries is the most common pathway to the root canal system for microbes. When the tooth is intact, enamel and dentin protect it against invasion of the pulp space. As caries approaches the

pulp, reparative dentin is laid down to avert exposure, but this rarely can prevent microbial entry without caries excavation. Dentinal tubules range from 1 to 4 m in diameter, whereas most bacteria are less than 1 m in diameter. Where the protective cementum layer is missing or if it has been lost through trauma, the dentinal tubules may be exposed and may serve as a pathway for microbial invasion of the pulp space. Bacterial movement is restricted by 1. outflow of dentinal fluid, 2. odontoblastic processes, 3. mineralized crystals, 4. And macromolecules, including immunoglobulins in the tubules. Bacteria and their byproducts may have an effect on the pulp before direct exposure. If the caries is removed, the pulp can heal. When a healthy vital pulp is exposed as a result of trauma, bacterial penetration of tissue proceeds relatively slowly. Bacteria penetrate less than 2 mm into pulp even after 2 weeks of exposure. However, if the pulp is necrotic, the "dead tracts" of empty dentinal tubules are quickly penetrated. Microbes rapidly reach the surface of the pulp after direct exposure of the pulp from restorative procedures, a traumatic injury, or an anomalous tooth development. The breakdown products of the necrotic pulp, serous exudate, and bacterial byproducts provide the nutrients for the invading organisms. Controversy still exists as to whether periodontal disease directly causes pulpal disease. Microbes and their byproducts may reach the pulp space through portals of entry at the apex of a root and through other lateral, accessory, or furcation canals.

Langeland et al found that pulpal necrosis occurred only when the apical
foramen was involved. For diagnostic purposes, it has been shown that abscesses of periodontal origin contain 30% to 58% spirochetes, whereas abscesses of endodontic origin contain fewer than 10% spirochetes; the microflora therefore does not closely resemble that of marginal periodontitis. Another possible route of entry is anachoresis, which is the transportation of microbes through the blood or lymph to an area of inflammation, such as a tooth with pulpitis. However, anachoresis may be one of the mechanisms by which some traumatized teeth become infected. Anachoresis could not be demonstrated in instrumented but unfilled canals.

Polymicrobial Endodontic Infections


In 1890 W.D. Miller, the father of oral microbiology, was the first investigator to associate the presence of bacteria with pulpal disease. A classic study published in 1965 by Kakehashi et al proved that bacteria caused pulpal and periradicular disease. These researchers found that exposure of the pulp in rats with normal microbial flora produced pulpal necrosis and the formation of periradicular lesions. However, no pathologic changes occurred in germ-free rats when the pulp was exposed. The germ-free rats healed with dentinal bridging regardless of the severity of the pulpal exposure, showing that the presence or absence of bacteria was the determinant for pulpal and periapical disease. Endodontic infections are polymicrobial. As culturing methods improved, the number of microorganisms detected in endodontic infections increased to a range of three to 12 organisms per infected root canal associated with an apical lesion. The number of colony forming units (CFU) is usually 102 to 108. A positive correlation exists between the number of bacteria in an infected root canal and the size of periradicular radiolucencies.
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Table 15-1. Bacteria from the Root Canals of Teeth with Apical Rarefactions
BACTERIA Fusobacterium nucleatum Streptococcus sp. Bacteroides sp.* Prevotella intermedia Peptostreptococcus micros Eubacterium alactolyticum Peptostreptococcus anaerobius Lactobacillus sp. Eubacterium lentum Fusobacterium sp. Campylobacter sp. Peptostreptococcus sp. Actinomyces sp. Eubacterium timidum Capnocytophaga ochracea Eubacterium brachy Selenomonas sputigena Veillonella parvula Porphyromonas endodontalis PERCENTAGE OF INCIDENCE 48 40 35 34 34 34 31 32 31 29 25 15 15 11 11 9 9 9 9

Prevotella buccae Prevotella oralis Propionibacterium propionicum Prevotella denticola Prevotella loescheii Eubacterium nodatum

9 8 8 6 6 6

*Nonpigmenting species Other species isolated in low incidence included Porphyromonas gingivalis, Bacteroides ureolyticus, Bacteroides gracilis, Lactobacillus minutus, Lactobacillus catenaforme, Enterococcus faecalis, Peptostreptococcus prevotii, Eienella corrodens, and Enterobacter agglomerans. (Adapted from Sundqvist: Taxonomy, ecology, and pathogenicity of the root canal, Oral Surg 78:522, 1994.)

Before 1970, because of the shortcomings of culturing methods, only a few strains of anaerobic bacteria were isolated from endodontic infections. Currently, most bacteria isolated from these infections are anaerobic. Table 15-1 lists the organisms most often cultivated from endodontic infections. This is a relatively small, restricted group of organisms compared with normal oral flora, which includes more than 500 species of cultivable bacteria. Through use of these methods, it is possible to compare the bacteria isolated from root canals to those isolated from the sulcus of a periodontal pocket. Cultivation of similar species of bacteria from the two sites indicates that the sulcus may be the source of bacteria in root canal infections. Strict anaerobes function at low oxidation-reduction potential and grow only in the absence of oxygen, but they vary in their sensitivity to oxygen. Obligate anaerobic bacteria lack the enzymes superoxide dismutase and catalase. Some species of bacteria are microaerophilic; they can grow in the presence of oxygen, but they derive most of their energy from anaerobic energy pathways. Facultative anaerobic bacteria can grow in the presence or absence of oxygen. Obligate aerobic bacteria have both superoxide dismutase and catalase and require oxygen for growth. The population of microflora in root canal infections is not static but rather changes over time. Microbial succession in root canals was studied in monkeys. Root canals in the animals were infected with indigenous oral bacteria and then sealed in the teeth for intervals of up to 1080 days. The results showed that a selective process takes place over time that allows anaerobic bacteria to predominate. After 1080 days, 98% of the bacteria cultured from the canals were strict anaerobes. Apparently, tissue fluid, necrotic pulp tissue, low oxygen tension, and bacterial byproducts determine which bacteria predominate. Some bacterial metabolites may be antagonistic to other bacteria. In addition, some species of bacteria produce bacteriocins,

which are proteins produced by one species that inhibit another species of bacteria. When the root canals of intact teeth with necrotic pulp were cultured in one study, strict anaerobes accounted for more than 90% of the bacteria. In another study, in which the apical 5 mm of carious exposed teeth was cultured, 67% of the bacteria were strict anaerobes. Apparently, then, the polymicrobial ecosystem in an infected root canal system selects for anaerobes. In fact, certain species tend to be associated with the presence of other species, which suggests a commensal relationship that is not random. Gram-negative bacteria, especially species of Porphyromonas and Prevotella that are dark (black) pigmented, have been associated with endodontic infections. Depending on the species and culture media, these colonies may vary from tan to black. Some species of dark-pigmented bacteria, peptostreptococci, peptococci, Fusobacterium spp., Eubacterium spp., and Actinomyces spp. have been implicated in certain clinical signs and symptoms. However, no absolute correlation has been established between any species of bacteria and the severity of endodontic infections. This is probably because the polymicrobial nature of endodontic infections and the synergistic relationship between bacteria or virulence factors increase the overall pathogenic effect. Polymicrobial infections spread from the root canal to the contiguous periradicular tissues. Endodontic abscesses are mixed infections involving several strains of bacteria. For example, studies have shown that strains of dark-pigmented bacteria in pure culture produce only a mild infection in an animal model; however, when these bacteria are mixed with another bacteria (e.g., Fusobacterium nucleatum), the combination produces abscesses and even death of the animals. Box 15-1 Recent Taxonomic Changes for Previous "Bacteroides" Species
Porphyromonas: Black-pigmented (asaccharolytic Bacteroides species) Porphyromonas asaccharolyticus (usually nonoral) Porphyromonas gingivalis*

Porphyromonas endodontalis* *Studies have associated species with clinical signs and symptoms. Prevotella: Black-pigmented (saccharolytic Bacteroides species) Prevotella melaninogenica Prevotella denticola

Prevotella loescheii Prevotella intermedia* Prevotella nigrescens Prevotella corporis

Prevotella tannerae Most commonly isolated species of black-pigmented bacteria from endodontic infections. Prevotella: Nonpigmented (saccharolytic Bacteroides species) Prevotella buccae* Prevotella bivia Prevotella oralis Prevotella oris Prevotella oulorum Prevotella ruminicola

Also, the current state-of-the-art culturing techniques may detect and identify only a portion of the total microbial population. Conventional identification of bacteria is based on 1. Gram staining, 2.colonial morphology, 3. Growth characteristics, and 4. Biochemical tests. Very often, microbial identification is only presumptive. Taxonomic revision based on deoxyribonucleic acid (DNA) studies has put species identification in older studies in question. For example, as a result of DNA methods, the dark-pigmented bacteria previously in the genus Bacteroides now have been placed in the genera Porphyromonas (asaccharolytic) and Prevotella (saccharolytic) (Box 15-1). Because of DNA methods, the species Prevotella nigrescens was separated from Prevotella intermedia. On the basis of this work, studies have shown that P. nigrescens is the darkpigmented bacteria most often cultivated from endodontic infections. DETECTION AND IDENTIFICATION OF PUTATIVE PATHOGENS

Microbial Cultivation and Molecular Diagnosis of Microbes


Cultivation relies on the growth of microorganisms in the laboratory under artificial growth conditions. This technique has some advantages because it 1. Allows identification of a great variety of microbial species in a sample, including those not being sought. 2. It also allows determination of the antimicrobial susceptibilities of the isolates. Cultivation methods have several important drawbacks : They are scarcely able or unable to grow many microorganisms; this is conducive to false-negative results and to underestimation of the pathogenic role played by some fastidious or uncultivable microorganisms. They often are slow to provide a diagnostic result (in anaerobic infections they are even too slow). They often have low specificity (because of ambiguity of

phenotypic features, the occurrence of convergent or divergent strains, and dependence on the experience of the microbiologist) and therefore have significant limitations in distinguishing among microbial species and strains. They have low sensitivity (failure to detect small numbers of microorganisms), particularly for fastidious anaerobic bacteria (103 to 104 cells). They strictly depend on the mode of sample transport, which must allow the survival of anaerobic bacteria but not favor overgrowth of facultative bacteria.

They are time-consuming, laborious, and expensive.

In the past two decades, methods based on molecular biology have been introduced for microbial identification. These methods rely on the fact that every living being, including microorganisms, has signature sequences in its genome that can be used as targets for precise identification and phylogenetic classification.Investigations of many aquatic and terrestrial environments using cultivation-independent (e.g., molecular) methods have revealed a great deal of previously unsuspected bacterial diversity. In fact, the cultivable members of these living systems represent less than 1% of the total population.

Novel culture-independent methods of microbial identification have also been used to determine the bacterial diversity associated with human diseased and healthy sites. Studies have shown that about 40% to 50% of the bacterial clones in the oral cavity and about 70% of clones in the gut and colon represent unknown and as yet uncultivated species. More than likely, then, some uncharacterized pathogens exist in this uncultivable proportion of the human microbiota. The question no longer is whether uncultivable microorganisms exist but why a huge proportion of the microbial species living in diverse environments cannot be grown under laboratory conditions. Microorganisms may be uncultivable for a number of reasons: 1. The artificial culture medium may lack the required nutrients or growth factors. Cultivation under laboratory conditions presupposes some knowledge of the microbial species' growth requirements. However, very little is known about the specific growth factors microorganisms use to survive in the human host. The culture medium itself may be toxic to some species and inhibit growth. 2. Other microorganisms in the sample may produce substances that inhibit the target species. 3. A species may depend on another species for growth. For example, Tannerella forsythia has an absolute requirement for N-acetyl muramic acid. It grows very poorly in pure culture but grows well in

coculture with other species. 4. Bacterial intercommunication may be disrupted, such as in quorum-sensing systems. Bacterial quorum-sensing systems, which may be important for coordinating the growth of component species in natural biofilms, are influenced by environmental conditions. Separation of bacteria on solid media is likely to disrupt such intercommunication and may help explain the incultivability of some species. Obviously, if microorganisms cannot be cultivated, they cannot be identified by phenotype-based methods. As long as researchers remain relatively ignorant about the needs of many bacteria for growth, identification methods are required that are not based on bacterial cultivability. Such methods prevent many pathogens from passing unnoticed in microbiologic surveys of clinical samples. Molecular diagnostic methods for microorganisms have several advantages over cultivation: 1. They can detect both cultivable and uncultivable microbial species and strains. 2. They have greater specificity and can accurately identify microbial strains with ambiguous phenotypic behavior, including divergent or convergent strains. 3. They can detect microbial species directly in clinical samples, without the need for cultivation. 4. They are more sensitive than other identification methods. In fact, the polymerase chain reaction (PCR) method much more sensitive than any other microbial diagnostic test. 5. They usually are less time consuming. 6. They do not require carefully controlled anaerobic conditions during sampling and transportation. If fastidious anaerobic bacteria and other fragile microorganisms lose viability during transit, they still can be identified. 7. They can be used during antimicrobial treatment. 8. When a large number of samples must be analyzed for epidemiologic studies, samples can be stored under low temperatures and surveyed all at once. Several methods based on molecular biology have been used to identify endodontic pathogens, and new techniques may be used in the near future. Many of the molecular methodologies are based on the use of the ribosomal RNA gene (rDNA) to identify microorganisms without the need for cultivation. The 16S rDNA gene is present in all bacteria; it has some regions that are virtually identical in all bacteria (conserved regions) and other regions that vary in sequence from one species to another (variable regions). Variable sequences in the 16S rDNA gene are unique signatures that allow specific identification.

DNA-DNA Hybridization Methodology DNA-DNA hybridization methods use DNA probes. In this process, segments of labeled, single-strand DNA locate and bind to their complementary nucleic acid sequences; after washing, the presence of bound label indicates the presence of the target DNA sequence. The DNA probe may target whole genomic DNA or individual genes (e.g., 16S rDNA). Whole genomic probes are more likely to cross-react with nontarget microor ganisms because of the presence of homologous sequences between different bacterial species. Oligonucleotide probes based on signature sequences of specific genes may show limited or no cross-reactivity with nontarget microorganisms. The checkerboard DNA-DNA hybridization method was introduced to allow hybridization of large numbers of DNA samples against large numbers of digoxigenin-labeled whole genomic DNA or 16S rRNA-based oligonucleotide probes on a single support nitrocellulose membrane.166 A MiniSlot device allows denatured DNA from clinical samples loaded in parallel channels to be aspirated through the membrane, depositing horizontal lanes on the membrane surface. Afterward, hybridizations are performed in vertical lanes with either digoxigenin-labeled whole genomic probes or oligonucleotide probes directly conjugated to alkaline phosphatase. This method allows simultaneous determination of the presence of a multitude of bacterial species in single or multiple clinical samples and is particularly useful for large-scale epidemiologic research. The detection limit of the checkerboard assay is approximately 103 to 104 cells.165,166 Polymerase Chain Reaction Method The molecular methods most often used for microbial identification are the PCR method and its variations. The PCR method involves in vitro replication of DNA and has often been referred to as the "genetic Xeroxing" approach. Repeated cycles of heating and cooling are used to make many copies of a specific region of DNA. First, the temperature is raised to near boiling, causing the double strand of DNA to denature into single strands. When the temperature is lowered, a pair of oligonucleotide primers (short DNA sequences) anneal to their complementary matches on the target DNA sequence. The primers define the limits of the DNA region to be copied. At a slightly higher temperature, the enzyme DNA polymerase binds to the primers and adds nucleotides to extend the second strand. In subsequent cycles, this process of denaturation, annealing, and extension is repeated to make additional DNA copies. Each new copy serves as a template for amplification in further cycles. After 30 cycles, millions of copies of the target sequence have been produced from a single starting molecule. PCR thus can create millions of identical copies of a target sequence in a matter of hours. This method has been hailed as one of the great breakthroughs in technology. PCR has unrivaled sensitivity; it can detect as few as 1 to 10 bacterial cells in a sample, making it at least 10 to 100 times more

sensitive than any other identification method. Moreover, PCR can have remarkable specificity because each distinct microbial species has unique DNA signature sequences. Genotypic traits remain fairly constant, offering a more reliable means of microbial identification. Numerous variations in the standard PCR procedure (here referred to as single PCR) have been developed. Several approaches for using PCR to detect microorganisms in clinical samples have been devised, each with its own advantages. The choice of a particular PCR approach depends on the questions addressed. Basically, PCR can be used to detect certain target species (species-specific PCR) or to identify virtually all bacterial species in a sample (broad-range PCR). In species-specific PCR, primers are designed that are complementary to unique stretches of DNA in the genome of the microbial species assayed. Single PCR, nested PCR, reverse transcriptase (RT)-PCR, multiplex PCR, and realtime PCR are examples of approaches commonly used to identify target species. Nested PCR uses the product of a primary PCR amplification as a template in a second PCR reaction. The first PCR products are subjected to a second round of amplification with another set of primers specific for an internal sequence that was amplified by the first primer set. This approach increases the sensitivity and can also increase specificity. Sensitivity is increased because the target DNA is amplified in the first round, with subsequent dilution of other DNA and inhibitors present in the sample. The use of another set of primers in the second round of amplification results in additional specificity. Even if nonspecific DNA amplification occurs in the first round, the nonspecific PCR product probably does not serve as a template in the second reaction because it is highly unlikely to have regions of DNA complementary to the second set of specific primers. The major drawback of the nested PCR protocol is the high probability of contamination during transfer of the firstround amplification products to a second reaction tube; therefore special precautions must be taken to prevent such contamination. RT-PCR was developed to amplify ribonucleic acid (RNA) targets. In RT-PCR, an RNA template is the initial target, and reverse transcriptase creates a complementary DNA (cDNA) copy of the RNA. Oligonucleotide primers catalyze conversion of a specific segment of RNA into DNA. Once the cDNA is formed, it can be used as a template for amplification as in standard PCR. This technique has the advantage of detecting messenger RNA (mRNA) encoding specific proteins and therefore provides important clues to the phenotype of the microorganism (e.g., the presence of mRNA encoding penicillinase provides important information about potential resistance to penicillin). Most PCR assays have focused on detection of a single pathogenic species by means of individual reactions. In multiplex PCR, two or more sets of

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primers specific for different targets are introduced in the same reaction tube. Thus more than one unique target sequence in a clinical specimen can be amplified at the same time. Multiplex PCR assays have been used to minimize the duration and expense of detection approaches. Most PCR assays are qualitative or can be adjusted to be semiquantitative. One exception is real-time PCR, which is characterized by continuous measurement of amplification products throughout the reaction. This method uses sequence-specific probes commonly associated with the detection of fluorescence resonance energy transfer. Two approaches have been frequently used: fluorescence emission caused by uncoupling of a reporter dye from a probe also carrying a quencher dye and fluorescence emission caused by bringing two dyes close together. By monitoring the release of fluorescence with each PCR cycle, the progress of the reaction is recorded in real time and the amount of DNA in the sample can be quantified. Real-time PCR assays allow the quantitation of individual target species and total bacteria in clinical samples. Also, assay time is significantly reduced because the need for postamplification detection of PCR products is eliminated. PCR can also be used to investigate microbial diversity in a given environment. In broad-range PCR, primers are designed that are complementary to conserved regions of a particular gene that are shared by a group of microorganisms. For example, primers complementary to conserved regions of 16S rDNA have been used with the intention of using the variable internal regions of the amplified sequence for sequencing and further identification. Broad-range PCR can detect the unexpected, and in this regard it is far more effective and accurate than culture techniques. Broadrange PCR has allowed the identification of several novel, fastidious, or uncultivable bacterial pathogens directly from diverse human sites.

PCR technology can be used for clonal analysis of microorganisms. Clonal analysis also may help to track the origin of microorganisms infecting a particular site. For example, comparison of bacterial strains isolated from the root canal and other oral sites may yield information about the origin of the microorganisms in the root canal system. In addition, clonal analysis can track the origin of a suspected focal disease through comparison of the clonal types found in the infected site with others present in other body sites, including the oral cavity (and infected root canals). As does any identification method, PCR methodology has drawbacks. Although PCR shows excellent detection limits, a relatively small amount of the sample is used in the amplification reaction. Consequently, if a given species is present in low numbers, the aliquot for PCR may not contain it. Clinical samples may contain enzyme inhibitors that may reduce or abort the amplification reaction. In addition, most PCR assays used for identification qualitatively detect the target microorganism but not its levels in the sample. Quantitative PCR assays (including real-time PCR) can resolve this problem.

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Most PCR assays detect only one species or a few different species (multiplex PCR) at a time. Broad-range PCR analysis overcomes this limitation, but it is laborious and expensive and may require multiple different reactions.

Molecular Methods to Detect Putative Pathogens


As mentioned earlier, molecular identification methods can overcome limitations imposed by a microorganism's cultivability. Studies using predominantly cultivation-dependent methods to identify bacterial species resulted in the establishment of a set of species thought to play an important role in the pathogenesis of periradicular lesions. More recently, molecular diagnostic techniques not only have confirmed these findings but have significantly expanded them. In other words, molecular methods have extended the data that support the role of many bacterial species in periradicular diseases and have also expanded the list of suspected endodontic pathogens by detecting species difficult or even impossible to cultivate. As a result, the known population of endodontic microbiota has been greatly expanded. Molecular diagnostic methods also have allowed the detection of some cultivable species in infected root canals or periradicular abscesses in higher prevalence values than ever reported by culturing studies. This has confirmed and even strengthened the association of those bacterial species with the etiology of periradicular diseases. Species that have been found in significantly higher prevalences with molecular methods include Porphyromonas endodontalis, Porphyromonas gingivalis, Fusobacterium nucleatum, Pseudoramibacter alactolyticus, Propionibacterium propionicum, Actinomyces spp., Slackia exigua, and Mogibacterium timidum (Fig. 15-1). Molecular methods can detect cultivable species at higher prevalences because of their greater sensitivity compared with cultivation, because of the fastidious nature of some microorganisms, or even because not all strains of a species can be cultivated. One study involving molecular analysis of purulent exudate aspirated from oral abscesses reported groups of uncultivable and previously uncharacterized bacteria that were predominant in samples. Moreover, the numbers of two cultivable species, F. nucleatum and P. endodontalis, were grossly underestimated in the samples. This showed that some cultivable species may include uncultivable strains. Molecular methods also have expanded the list of putative endodontic pathogens by including some fastidious bacterial species or even uncultivable bacteria that had not been detected in endodontic infections by cultivation procedures. The important periodontal

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pathogen T. forsythia (formerly Bacteroides forsythus) had never been detected in infected root canals by culture. Recent studies using diverse molecular techniques have shown that T. forsythia is a common member of the microbiota associated with different types of endodontic infections, including endodontic abscesses (Fig. 15-2). Depending on the molecular identification approach used, prevalence values for T. forsythia have ranged from 18% to 52% of the cases investigated.* Although spirochetes frequently had been observed by microscopy in samples taken from endodontic infections, they had never been identified. Molecular diagnostic methods used to identify spirochetes have shown that these spiral bacteria were overlooked because of technical hurdles posed by culturing approaches. Molecular studies have demonstrated a high prevalence of Treponema denticola, a recognized periodontal pathogen, in infected root canals and abscesses. T. denticola also has been found in endodontic infections in association with T. forsythia and P. gingivalis. These three pathogens comprise the red complex, which is implicated in severe forms of periodontal diseases. PCR methods also have identified Treponema socranskii in a high number of root canal infections. Other treponemes, such as Treponema lecithinolyticum, Treponema vincentii, Treponema pectinovorum, Treponema maltophilum, Treponema amylovorum, and Treponema medium, have only recently been demonstrated in endodontic infections. Other new species included by molecular investigations in the restricted set of putative endodontic pathogens include Prevotella tannerae, Dialister pneumosintes, Filifactor alocis, Fusobacterium periodonticum, Olsenella spp., Eubacterium infirmum, and Centipeda periodontii (Fig. 15-1).

Investigations of the diversity of the endodontic microbiota by broad-range PCR followed by cloning and sequencing have demonstrated that it is far more complex than previously anticipated. One study assessed the diversity of bacteria present in infected root canals. Two primarily infected root canals yielded sequences related to those of the genera Enterococcus, Lactobacillus, Propionibacterium, and Streptococcus, and two clones were related to previously uncultivated bacteria. These researchers concluded that molecular techniques may detect bacteria in endodontic infections when culture techniques yield a negative result and thus can be used to identify a

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wider range of endodontic bacteria, including previously unidentified or uncultivable bacteria. Another study compared the results from cultural and molecular analyses (broad-range PCR, cloning, and sequencing) of microorganisms in samples taken from five infected root canals.120 Sixty-five taxa were identified, of which 26 (40%) were uncultivable and therefore not detected by culture. A mean of 20 taxa was found in each sample. A new species of Dialister was present in all examined cases.

Geographic Influence on Endodontic Microorganisms Findings from laboratories in different countries often differ considerably regarding the prevalence of the species involved in endodontic infections. Although these differences may be attributed to variations in the identification methodologies, a geographic influence in the composition of the root canal microbiota is suspected. Molecular methods detect DNA, which can remain relatively unaltered and therefore detectable for a long time when stored under proper conditions. Therefore samples can be submitted all at once, and loss of detectability because of transportation issues can be prevented. One study used species-specific PCR to detect selected bacterial species in abscess samples collected from two distant geographic locations. The study showed a significant difference in the prevalence of several species, which suggests that geographic factors may influence the composition of the endodontic microbiota.

Failed Endodontic Treatment


Failure of a periradicular lesion to heal after endodontic treatment likely is often related to microbes remaining in the root canal system that have access to periradicular tissues. Recent studies of endodontically treated teeth requiring retreatment have shown a prevalence of facultative Gram-positive bacteria in low numbers in the root canals. In contrast, high numbers of anaerobic Gram-negative bacteria are found in primary root canal infections.

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Enterococcus faecalis has been the predominant microbe in canals undergoing retreatment. In one study, complete periapical healing occurred in 94% of roots with negative culture results at the obturation appointment, whereas complete healing occurred in just 68% of cases with positive culture results at the time of obturation. These results supported previous studies that showed that failure to heal is more likely when canals are obturated in the presence of a persisting infection. PCR has been used to study microorganisms in cases of failed endodontic therapy. Using broad-range PCR, one study reported that five refractory cases produced clones related to the genera Capnocytophaga, Cytophaga, Dialister, Eubacterium, Fusobacterium, Gemella, Mogibacterium, Peptostreptococcus, Prevotella, Propionibacterium, Selenomonas, Solobacterium, Streptococcus, and Veillonella, as well as two clones representing previously uncultivated bacteria. Another recent study used single PCR to investigate the occurrence of several microbial species in cases of failed endodontic therapy; E. faecalis was found in 77% of cases, confirming that this microbe is the most prevalent species in failed endodontic treatment. Four other fastidious anaerobic species, P. alactolyticus, P. propionicum, F. alocis, and D. pneumosintes, were present in half of the failed cases. All examined cases harbored at least one of the Gram-positive bacterial species E. faecalis, P. alactolyticus, or P. propionicum. Two studies have used the checkerboard DNA-DNA hybridization technique to identify bacteria in periradicular lesions of asymptomatic teeth. Both studies identified bacterial DNA in all samples. Detected species included P. endodontalis, F. nucleatum, P. intermedia, T. denticola, Actinobacillus actinomycetemcomitans, and T. forsythia. The results of both studies indicated that bacteria may invade periradicular lesions. Many more bacteria were detected with the checkerboard DNADNA hybridization method than had previously been recovered by anaerobic culture.

Diversity of Endodontic Microorganisms


Although bacteria are by far the most common microorganisms involved in endodontic infections, studies have revealed a possible role for fungi and more recently for viruses. Fungi are eukaryotic microorganisms that have been detected by culture in endodontic infections. Studies of samples collected from primary root canal infections have shown a range of findings, from as few as 2% of samples with fungal DNA to as many as 21% with DNA from Candida albicans.

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C. albicans has been reported in 9% of samples collected from failed endodontic cases. The recent use of molecular methods to detect herpes viruses in periradicular lesions has suggested that some herpes viruses can participate in the etiology of periradicular lesions. Sabeti et al used a RT-PCR approach to compare the presence of late transcripts of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) in samples taken from symptomatic and asymptomatic periradicular lesions at the time of periradicular surgery. HCMV was detected in seven of seven symptomatic lesions and in one of seven asymptomatic lesions. EBV was found in six of seven symptomatic lesions and in one of seven asymptomatic lesions. One asymptomatic lesion was positive for HSV. These data suggest that HCMV and EBV present in active infections can participate in the pathogenesis of symptomatic periradicular lesions. A higher occurrence of herpes viruses has also been observed in large periradicular lesions compared with small ones. This study also suggested that an association between herpes viruses and endodontic bacterial pathogens may play a role in causing symptomatic periradicular lesions. Molecular methods have been used to analyze clonal diversity within certain endodontic bacteria. Using both ERIC-PCR and AP-PCR approaches, one study reported the occurrence of different clonal types of F. nucleatum coexisting in infected root canals. The possible existence of pathogenic and nonpathogenic clonal types of F. nucleatum might explain why F. nucleatum strains are isolated and detected in high prevalence in both symptomatic and asymptomatic endodontic infections. Another study used AP-PCR to examine the genetic diversity of the isolates of P. gingivalis or P. nigrescens from infected root canals and from subgingival plaque.This study revealed that the clonal types simultaneously present in the root canal system and in subgingival plaque of all patients were genotypically indistinguishable, which suggests that the subgingival plaque can be a source of root canal microorganisms.

A high proportion of the bacterial species inhabiting the oral cavity is still uncultivable, and this proportion likely includes previously unrecognized pathogens. Further molecular studies, aided by advances in molecular technology, may allow discovery of novel pathogens, may further strengthen the causal relationships between specific bacteria and certain oral diseases, and may shed light on the role of viruses in

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the pathogenesis of periradicular disease. In the near future, the clinical microbiology laboratory will look like a molecular biology laboratory. PCR and other molecular biology techniques offer the hope of refining our knowledge of endodontic infectious processes and of making rapid diagnosis and directed antimicrobial therapy a reality. BACTERIAL VIRULENCE FACTORS Although the initial injury may be associated with other causes (e.g., physical or chemical trauma), most pathogenic responses are associated with microorganisms and associated host responses. Microbes have numerous virulence factors, including bacterial capsules, fimbriae (pili), lipopolysaccharides (LPS), enzymes, extracellular vesicles, fatty acids, polyamines, ammonia, and hydrogen sulfide. Both Gram-positive and Gram-negative bacteria have capsules that may protect the microbe from phagocytosis. Fimbriae and extracellular vesicles may participate in aggregation of bacteria or attachment to tissues. Pili may extend from one bacterium to another during conjugation and exchange DNA for virulence factors, including resistance to antibiotics. LPS, when released from the outer membrane of Gram-negative bacteria, it is called endotoxin. Endotoxin has several biologic effects, including the activation of complement and bone resorption. Studies have shown that the concentration of endotoxin in the canals of symptomatic teeth is higher than that in the canals of asymptomatic teeth. Enzymes are produced by bacteria and are detrimental to the host. A recent study showed that the gene for collagenase , a metalloproteinase associated with the spread of cellulitis could be detected in strains of P. gingivalis but not P. endodontalis isolated from endodontic infections. Other enzymes produced by bacteria neutralize immunoglobulins and the components of complement. In abscesses, neutrophils lyse and release their enzymes into the surrounding milieu, forming a purulent exudate. This enzyme-rich exudate has an adverse affect on the surrounding tissues.

Extracellular vesicles are formed from the outer membrane of Gram-negative bacteria and have a trilaminar structure similar to that of the parent bacteria. Because they have the same surface antigens, they can bind antibodies directed against the parent organism and thus reduce the host's humoral response to infection. The vesicles may contain enzymes or other toxic agents. Extracellular vesicles are

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believed to be involved in hemagglutination, hemolysis, bacterial adhesion, and proteolytic action on host tissues. The short chain fatty acids most commonly produced by bacteria in infected root canals are propionic, butyric, and isobutyric acids. Short chain fatty acids affect neutrophil chemotaxis, degranulation, chemiluminescence, and phagocytosis. Butyric acid exerts the greatest inhibition of T-cell blastogenesis and stimulates the production of interleukin-1 (IL-1), which is associated with bone resorption Polyamines are biologically active compounds involved in the regulation of growth, regeneration of tissues, and modulation of inflammation. They include spermine, spermidine, cadaverine, and putrescine. Polyamines are produced by both bacteria and host cells and are found in infected root canals. Teeth that are painful to percussion or have spontaneous pain have been shown to have a higher concentration of total polyamines in necrotic pulp. Microbial coaggregation or self-aggregation similar to dental plaque may protect the bacteria and act as a virulence factor. The combination of different species of bacteria was shown to be more virulent in mice than the organisms in pure culture. This supports the belief that additive and synergistic relationships between the organisms in polymicrobial infections, such as in endodontics, may increase the overall pathogenicity. Although numerous species of bacteria have been associated with clinical signs and symptoms, no absolute correlation has been made with any specific species. Studies have shown that the efficacy of various irrigants and intracanal medicaments was related to the nature of the organisms in a biofilm and to contact time.

EXTRARADICULAR ENDODONTIC INFECTIONS


Most host immune cells associated with an untreated, infected root canal are T lymphocytes. During the first 15 days of the development of a periapical lesion in a rat model, the T-helper cells outnumbered the T-suppressor cells. However, after 15 days the T-suppressor cells outnumbered the T-helper cells. The T-helper cells seemed to be associated with bone resorption and lesion expansion. In another study, the cells associated with endodontically treated teeth had more B lymphocytes than T lymphocytes. This shows that the periradicular inflammatory tissue can mount an immunologic response to bacteria and bacterial byproducts. Several studies using enzyme-linked immunosorbent assay (ELISA), radioimmunosorbent tests, and radial immunodiffusion assays have demonstrated the presence of IgG, IgA, IgM, or IgE in periapical lesions In

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one study, IgG produced from human periapical explants was more often reactive with dark-pigmented bacteria (e.g., P. intermedia, P. endodontalis, and P. gingivalis) than any of the other species of bacteria tested. In addition, an increase in the level of serum IgG reactive with P. intermedia has been shown to be associated with the existence of periodontal disease or combined endodontic and periodontal disease. Another investigation showed that samples from root canals associated with symptomatic periapical lesions contained elevated amounts of beta-glucuronidase and interleukin-1-beta. IL-1 and prostaglandins seem to be especially associated with bone resorption. However, other mediators, such as kinins and neuropeptides, also are involved in the inflammatory response. The antigens of bacteria and bacterial byproducts stimulate both B cells and T cells. For example, LPS can cause both polyclonal stimulation of B cells and macrophage activation. The teleologic purpose of chronic periradicular lesions (i.e., periapical granulomas) is believed to be prevention of the spread of infection to surrounding tissues. As some researchers have put it, "a granuloma is not an area in which bacteria live but in which they are destroyed." Investigators have cultured bacteria from asymptomatic, chronic periradicular lesions. Microbial culture contaminants are a concern with surgical procedures or direct communication (e.g., from sinus tracts) or from the root apex during curettage of the extraradicular sample. However, as evidenced by the presence of periapical abscesses, bacteria do invade periradicular tissues. Prior to formation of an abscess or cellulitis or both, there would be some point in time when microbial invasion takes place before a symptomatic inflammatory response is mounted against the invading organisms. Nair used light and electron microscopy to observe intracellular and extracellular microorganisms in four symptomatic granulomas and one asymptomatic cyst. In 25 other teeth with asymptomatic, chronic inflammatory lesions, bacteria could not be identified beyond the root apex. Species of Actinomyces and Propionibacterium have been shown to persist in inflamed tissue. Actinomyces israelii, a species of bacteria isolated from periapical tissues, does not always respond to conventional endodontic therapy, but both sodium hypochlorite and calcium hydroxide have proved highly effective at killing A. israelii. Endodontic surgery apparently is an effective means of removing A. israelii from the periapex and seems to achieve a high success rate without the use of antibiotics.

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However, when a periradicular infection with A. israelii is not resolved by surgery, antibiotic therapy is optimized by the use of amoxicillin or cephalexin . This extended regimen of antibiotics is necessary in only a few cases. The presence of A. israelii should be confirmed by either a biopsy or a culture showing the presence of Actinomyces sp. when a lesion does not heal after conventional treatment. Studies have detected fungi after treatment of therapy-resistant endodontic problems. One study showed that strains of C. albicans required incubation with a saturated solution of calcium hydroxide for about 16 hours to kill 99.9% of the fungi. Another study evaluated samples of tissue from cases of therapy-resistant, chronic apical periodontitis and identified 48 fungi in 7% of the samples (47 of 692 samples). A third study, using scanning electron microscopy (SEM), found fungi in the dentinal tubules of four of 10 extracted human molars with infected root canals. In a molecular study, PCR detected C. albicans in five of 24 intact teeth with infected root canals (but not in 19 samples aspirated from periradicular abscesses or cellulitic tissue). Abscesses and cellulitis develop when bacteria invade and infect periradicular tissues. Chemotaxis of neutrophils is a nonspecific inflammatory response to the presence of bacteria in normally sterile tissues. With accumulation of neutrophils and the resulting purulent exudate, an acute apical inflammatory response develops. The severity of the infection depends on the number and virulence of the bacteria, host resistance, and associated anatomic structures. An abscess is an accumulation of purulent exudate consisting of bacteria, bacterial byproducts, inflammatory cells (mainly neutrophils), lysed inflammatory cells, and the contents of those cells (e.g., enzymes). Cellulitis is a diffuse, erythematous, mucosal, or cutaneous infection that may spread to deeper facial spaces and become life threatening. Needle aspirates often reveal pockets of pus in a diffuse cellulitis. From a clinical viewpoint, cellulitis and abscesses may be considered a continuum of the inflammatory process. It is just a matter of time before purulence becomes visible in an area of cellulitis. FASCIAL SPACE INFECTIONS

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If bacteria from the infected pulp tissue gain entry into the periradicular tissue and the immune system is unable to suppress the invasion, an otherwise healthy patient eventually shows signs and symptoms of an acute periradicular abscess, cellulitis, or both. Clinically, the patient experiences swelling and mild to severe pain. Depending on the relationship of the apices of the involved tooth to the muscular attachments, the swelling may be localized to the vestibule or may extend into a fascial space. The patient may also have systemic manifestations, such as fever, chills, lymphadenopathy, headache, and nausea. Because the reaction to the infection may occur very quickly, the involved tooth may or may not show radiographic evidence of a widened periodontal ligament space. However, in most cases the tooth elicits a positive response to percussion, and the periradicular area is tender to palpation. In such cases the tooth is serving as a focus of infection because it leads to periapical infection and secondary (i.e., metastatic) spread to the fascial spaces of the head and neck (resulting in cellulitis and systemic signs and symptoms of infection). In most cases treatment involves - incision for drainage and root canal treatment of the involved tooth to remove the source of the infection. -Antibiotic therapy may be indicated with compromised host resistance, the presence of systemic symptoms, or fascial space involvement. Fascial space infections of odontogenic origin are infections that have spread into the fascial spaces from the periapical area of a tooth, the focus of infection. They are not examples of the theory of focal infection, which describes the dissemination of bacteria or their toxic products from a distant focus of infection. Fascial spaces are potential anatomic areas that exist between the fascia and underlying organs and other tissues. During an infection, these spaces are formed as a result of the spread of purulent exudate. The spread of infections of odontogenic origin into the fascial spaces of the head and neck is determined by the location of the root end of the involved tooth in relation to its overlying buccal or lingual cortical plate and the relationship of the apex to the attachment of a muscle. For example, if the source of the infection is a mandibular molar and the apices of the molar lie closer to the lingual cortical plate and above the attachment of the mylohyoid muscle of the floor of the mouth, the purulent exudate breaks through the lingual cortical plate into the sublingual space

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If the apices lie below the attachment of the mylohyoid muscle, the infection spreads into the submandibular space. As described by Hohl et al, the fascial spaces of the head and neck can be categorized into four anatomic groups:

The mandible and below The cheek and lateral face The pharyngeal and cervical areas The midface

Swellings of and below the mandible include six anatomic areas or fascial spaces:

The buccal vestibule The body of the mandible The mental space The submental space The sublingual space The submandibular space

The mandibular buccal vestibule is the anatomic area between the buccal cortical plate, the overlying alveolar mucosa, and the buccinator muscle (in the posterior) or the mentalis muscle (in the anterior) In this case the source of the infection is a mandibular posterior or anterior tooth in which the purulent exudate breaks through the buccal cortical plate and the apex or apices of the involved tooth lie above the attachment of the buccinator or mentalis muscle, respectively. The space of the body of the mandible is the potential anatomic area between the buccal or lingual cortical plate and its overlying periosteum. The source of infection is a mandibular tooth in which the purulent exudate has broken through the overlying cortical plate but not yet perforated the overlying periosteum. Involvement of this space can also occur as a result of a postsurgical infection. The mental space is the potential bilateral, anatomic area of the chin that lies between the mentalis muscle superiorly and the platysma muscle inferiorly. The source of the infection is an anterior tooth in which the purulent exudate breaks through the buccal cortical plate and the apex of the tooth lies below the attachment of the mentalis muscle. The submental space is the potential anatomic area between the mylohyoid muscle superiorly and the platysma muscle inferiorly. The source of the infection is an anterior tooth in which the purulent exudate breaks through the lingual cortical plate and the apex of the tooth lies below the attachment of the mylohyoid muscle. 22

The sublingual space is the potential anatomic area between the oral mucosa of the floor of the mouth superiorly and the mylohyoid muscle inferiorly. The lateral boundaries of the space are the lingual surfaces of the mandible. The source of infection is any mandibular tooth in which the purulent exudate breaks through the lingual cortical plate and the apex or apices of the tooth lie above the attachment of the mylohyoid muscle. The submandibular space is the potential space between the mylohyoid muscle superiorly and the platysma muscle inferiorly. The source of infection is a posterior tooth, usually a molar, in which the purulent exudate breaks through the lingual cortical plate and the apices of the tooth lie below the attachment of the mylohyoid muscle. If the submental, sublingual, and submandibular spaces are involved at the same time, a diagnosis of Ludwig's angina is made. This life-threatening cellulitis can advance into the pharyngeal and cervical spaces, resulting in airway obstruction. Swellings of the lateral face and cheek include four anatomic areas or fascial spaces:

The buccal vestibule of the maxilla The buccal space The submasseteric space The temporal space

The buccal vestibular space is the area between the buccal cortical plate, the overlying mucosa, and the buccinator muscle. The superior extent of the space is the attachment of the buccinator muscle to the zygomatic process. The source of infection is a maxillary posterior tooth in which the purulent exudate breaks through the buccal cortical plate and the apex of the tooth lies below the attachment of the buccinator muscle. The buccal space is the potential space between the lateral surface of the buccinator muscle and the medial surface of the skin of the cheek. The superior extent of the space is the attachment of the buccinator muscle to the zygomatic arch, whereas the inferior and posterior boundaries are the attachment of the buccinator to the inferior border of the mandible and the anterior margin of the masseter muscle, respectively. The source of the infection can be either a posterior mandibular or maxillary tooth in which the purulent exudate breaks through the buccal cortical plate and the apex or apices of the tooth lie above the attachment of the buccinator muscle (i.e., maxilla) or below the attachment of the buccinator muscle (i.e., mandible). The submasseteric space is the potential space between the lateral surface of the ramus of the mandible and the medial surface of

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the masseter muscle. The source of the infection is usually an impacted third molar in which the purulent exudate breaks through the lingual cortical plate and the apices of the tooth lie very close to or within the space. The temporal space is divided into two compartments by the temporalis muscle. The deep temporal space is the potential space between the lateral surface of the skull and the medial surface of the temporalis muscle; the superficial temporal space lies between the temporalis muscle and its overlying fascia. The deep or superficial temporal spaces are involved indirectly if an infection spreads superiorly from the inferior pterygomandibular or submasseteric spaces, respectively. Swellings of the pharyngeal and cervical areas include the following fascial spaces:

The pterygomandibular space The parapharyngeal spaces The cervical spaces

The pterygomandibular space is the potential space between the lateral surface of the medial pterygoid muscle and the medial surface of the ramus of the mandible. The superior extent of the space is the lateral pterygoid muscle. The source of the infection is mandibular second or third molars in which the purulent exudate drains directly into the space. In addition, contaminated inferior alveolar nerve injections can lead to infection of the space. The parapharyngeal spaces comprise the lateral pharyngeal and retropharyngeal spaces .The lateral pharyngeal space is bilateral and lies between the lateral surface of the medial pterygoid muscle and the posterior surface of the superior constrictor muscle. The superior and inferior margins of the space are the base of the skull and the hyoid bone, respectively, and the posterior margin is the carotid space, or sheath, which contains the common carotid artery, internal jugular vein, and the vagus nerve. Anatomically, the retropharyngeal space lies between the anterior surface of the prevertebral fascia and the posterior surface of the superior constrictor muscle and extends inferiorly into the retroesophageal space, which extends into the posterior compartment of the mediastinum. The pharyngeal spaces usually become involved as a result of secondary spread of infection from other fascial spaces or directly from a peritonsillar abscess. The cervical spaces comprise the pretracheal, retrovisceral, danger, and prevertebral spaces. The pretracheal space is the potential space surrounding the trachea. It extends from the thyroid cartilage inferiorly into the superior portion of the anterior compartment of the mediastinum to the level of

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the aortic arch. Because of its anatomic location, odontogenic infections do not spread to the pretracheal space. The retrovisceral space comprises the retropharyngeal space superiorly and the retroesophageal space inferiorly. The space extends from the base of the skull into the posterior compartment of the mediastinum to a level between vertebrae C6 and T4. The danger space (i.e., space 4) is the potential space between the alar and prevertebral fascia. Because this space is composed of loose connective tissue, it is considered an actual anatomic space extending from the base of the skull into the posterior compartment of the mediastinum to a level corresponding to the diaphragm. The prevertebral space is the potential space surrounding the vertebral column. As such, it extends from vertebra C1 to the coccyx. A retrospective study showed that 71% of cases in which the mediastinum was involved arose from the spread of infection from the retrovisceral space (21% from the carotid space and 8% from the pretracheal space). Swellings of the midface consist of four anatomic areas and spaces:

The palate The base of the upper lip The canine spaces The periorbital spaces

Odontogenic infections can spread into the areas between the palate and its overlying periosteum and mucosa and the base of the upper lip, which lies superior to the orbicularis oris muscle, even though these areas are not considered actual fascial spaces. The source of infection of the palate is any of the maxillary teeth in which the apex of the involved tooth lies close to the palate. The source of infection of the base of the upper lip is a maxillary central incisor in which the apex lies close to the buccal cortical plate and above the attachment of the orbicularis oris muscle. The canine, or infraorbital, space is the potential space between the levator anguli oris muscle inferiorly and the levator labii superioris muscle superiorly. The source of infection is the maxillary canine or first premolar in which the purulent exudate breaks through the buccal cortical plate and the apex of the tooth lies above the attachment of the levator anguli oris muscle. The periorbital space is the potential space that lies deep to the orbicularis oculi muscle. This space becomes involved through the spread of infection from the canine or buccal spaces. Infections of the midface can be very dangerous because they can result in cavernous sinus thrombosis, a life-threatening infection in which a thrombus formed in the cavernous sinus breaks free, resulting in blockage of an artery or the spread of infection.

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Under normal conditions, the angular and ophthalmic veins and the pterygoid plexus of veins flow into the facial and external jugular veins. If an infection has spread into the midfacial area, however, edema and resultant increased pressure from the inflammatory response cause the blood to back up into the cavernous sinus. Once in the sinus, the blood stagnates and clots. The resultant infected thrombi remain in the cavernous sinus or escape into the circulation.

MANAGEMENT OF ABSCESSES AND CELLULITIS


The two most important elements of effective patient management are correct diagnosis and removal of the cause of an endodontic infection. In an otherwise healthy patient, chemomechanical debridement of the infected root canal (or canals) and incision for drainage of periradicular swelling usually prompt rapid improvement in clinical signs and symptoms. Most endodontic infections can be treated effectively without the use of adjunctive antibiotics. The appropriate treatment is removal of the cause of the inflammatory condition. Antibiotics (i.e., antimicrobials) are not recommended for irreversible pulpitis, acute apical periodontitis, a draining sinus tract, after endodontic surgery, to prevent flare-ups, or after incision for drainage of a localized swelling (without cellulitis, fever, or lymphadenopathy). In these situations, when the ratio of risk to benefit is considered, antibiotic use may put the patient at risk for the side effects of the antimicrobial agent and select for resistant organisms. Analgesics (not antibiotics) are indicated for the treatment of pain. Antibiotics are recommended, in conjunction with appropriate endodontic treatment, for progressive or persistent infections with systemic signs and symptoms such as fever (100F [37.8C]), malaise, cellulitis, unexplained trismus, and progressive or persistent swelling (or both). Antibiotic therapy is indicated as an adjunct to effective debridement of the root canal system, which is the reservoir of the organisms. In addition, aggressive incision for drainage is indicated for any infection marked by cellulitis. Incision for drainage is indicated whether the cellulitis is indurated or fluctuant. It is important to provide a pathway of drainage to prevent further spread of the abscess and/or cellulitis. An incision for drainage allows decompression of the increased tissue pressure associated with edema and provides significant pain relief. Furthermore, the incision provides a pathway not only for bacteria and bacterial byproducts but also for the inflammatory mediators associated with the spread of cellulitis. A minimum inhibitory concentration of antibiotic may not reach the source of the infection because of decreased blood flow and

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because the antibiotic must diffuse through the edematous fluid and pus. Drainage of edematous fluid and purulent exudate improves circulation to the tissues associated with an abscess or cellulitis, providing better delivery of the antibiotic to the area. Placement of a drain may not be indicated for localized fluctuant swellings if complete evacuation of the purulent exudate is believed to have occurred. For effective drainage, a stab incision is made in the most dependent site of the swelling through the periosteum. The incision must be long enough to allow blunt dissection using a curved hemostat or periosteal elevator under the periosteum for drainage of pockets of inflammatory exudate. A rubber dam drain or a Penrose drain is indicated for any patient with a progressive abscess or cellulitis to maintain an open pathway for drainage. Patients should improve rapidly after removal of the cause of the infection and drainage. Patients with cellulitis should be followed on a daily basis to ensure that the infection is resolving. Endodontic treatment should be completed as soon as possible after the incision for drainage. The drain usually can be removed 1 or 2 days after improvement is noted in clinical signs and symptoms. If no significant improvement occurs, the diagnosis and treatment must be reviewed carefully. Consultation with a specialist and referral may be indicated for severe infections or persistent infections. Likewise, patients requiring extraoral drainage should be referred to a clinician trained in the technique.

ANTIBIOTICS FOR ENDODONTIC INFECTIONS


Ideally, susceptibility testing would be done when antibiotics are indicated. However, antibiotics often are prescribed empirically because testing on strict anaerobes may take several days to weeks, and many organisms are not cultivable. Empiric prescription of antibiotics is based on a knowledge of the organisms most likely associated with endodontic infections. Clinicians should inform patients of the benefits, risks, and side effects of a drug, as well as the problems that can arise if the proper dosing schedule is not followed. Although the antitubercular drug rifampin seems to be the only antimicrobial proven to reduce the effectiveness of oral contraceptives, case reports have implicated other antibiotics.\ Patients using oral contraception, therefore, should be warned to use alternative methods

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of birth control. Typical regimen for treating an endodontic infection is 6 to 10 days on an around-the-clock schedule. Improvement should be seen in 24 to 48 hours after initial treatment and initiation of the prescription. A high-dose regimen for a short time is preferred to a low dose for a longer time. The latter approach is much more likely to select for resistant organisms. A loading dose generally is recommended to provide an initial effective serum level; the loading dose is followed by the maintenance dosage. Penicillin VK has a spectrum of microbial activity that includes many of the bacteria most often associated with endodontic infections, including both facultative and anaerobic bacteria. Penicillin VK remains the antibiotic of choice for treatment of endodontic infections because of its efficacy and low toxicity. However, all the penicillins have up to a 10% allergy rate, which is a major concern. An oral loading dose of 1000 mg should be followed by 500 mg every 6 hours for 6 to 10 days. For severe infections the antibiotic may be taken every 4 hours to maintain a more constant serum level. Amoxicillin has a broader spectrum of activity than penicillin VK that includes bacteria usually not isolated from endodontic infections. It is absorbed more rapidly and gives a higher and more sustained serum level. However, because of its wider spectrum, it selects for more resistant organisms, especially in the gastrointestinal tract. For patients who are immunocompromised or otherwise medically compromised, prescription of a broader spectrum penicillin may be warranted. An oral loading dose of 1000 mg of amoxicillin should be followed by 500 mg every 8 hours for 6 to 10 days. The combination of amoxicillin with clavulanate (i.e., Augmentin) recently was shown to have the best efficacy against bacteria isolated from endodontic infections and may be indicated to treat serious endodontic infections, especially in immunocompromised patients. Clarithromycin and azithromycin are macrolides (like erythromycin ) but are effective against some of the anaerobic species of bacteria associated with endodontic infections (unlike erythromycin ). These antimicrobials do not have a long-term track record but may be considered for mild infections in patients allergic to penicillin. They produce less gastrointestinal upset than erythromycin . Clarithromycin may be given with or without meals in a dose of 250 to 500 mg every 12 hours for 6 to 10 days. Azithromycin should be taken 1 hour before meals or 2 hours after meals, at a loading dose of 500 mg on the first day, followed by 250 mg daily. These antimicrobials block the metabolism of a number of

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drugs; therefore care must be taken to check for interaction with other drugs the patient may be taking. For example, these antimicrobials can block the metabolism of warfarin and anisindione , which can lead to serious bleeding in patients undergoing anticoagulation therapy. Metronidazole is active against strict anaerobes but ineffective against facultative bacteria. It can be used with penicillin when the latter has been used for 2 to 3 days without improvement in the patient's signs and symptoms. The addition of metronidazole to penicillin is indicated after the diagnosis has been reviewed and appropriate drainage has been performed. Metronidazole is prescribed in a loading dose of 500 mg, followed by 250 to 500 mg every 6 hours. Penicillin should be continued because metronidazole is not effective against facultative bacteria. Patients taking metronidazole should not consume alcohol during therapy and for at least 3 days afterward because of a disulfiram type of reaction. Likewise, metronidazole should not be prescribed for patients taking lithium . Clindamycin is recommended for patients with a serious infection who are allergic to penicillin. It is effective against both facultative and strict anaerobes. Clindamycin is distributed throughout the body and concentrates in bone. Although antibiotic-associated colitis (i.e., pseudomembranous colitis) has been linked to clindamycin , it occurs only rarely at the dosages recommended for endodontic infections. In addition, antibiotic-associated colitis has been associated with numerous other antibiotics, although not the aminoglycosides. Clindamycin should be prescribed at a loading dose of 300 mg, followed by 150 to 300 mg every 6 hours for 6 to 10 days.

PROPHYLACTIC ANTIBIOTICS FOR MEDICALLY COMPROMISED PATIENTS The American Heart Association (AHA) has updated its guidelines for prophylactic antibiotic coverage for medically compromised patients. The guidelines are based not on controlled clinical studies but on an analysis of relevant articles. The guidelines are published as an aid to clinicians and not intended as a standard of care or as a substitute for clinical judgment.

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If any question exists about the need for antibiotic prophylaxis, the clinician should request a consultation with the patient's physician. The incidence and load of bacteremia is relatively low for nonsurgical root canal therapy, but microorganisms can be extruded past the root apex. A population-based study concluded that dental treatments, in general, did not seem to be a risk factor for infective endocarditis. If more studies support these findings, future consideration should be given to downgrading antibiotic prophylaxis for most dental procedures other than tooth extractions and gingival surgery. For high-risk patients, the AHA recommends antibiotic endocarditis prophylaxis for root canal instrumentation or surgery beyond the root apex and for intraligamentary injections of a local anesthetic. Endocarditis prophylaxis is not recommended for nonintraligamentary local anesthetic injections, rubber dam placement, or the taking of radiographs. For patients who are not allergic to penicillins, amoxicillin in a dose of 2 g 1 hour before the dental procedure is recommended for antibiotic prophylaxis. Amoxicillin is recommended because it is better absorbed from the gastrointestinal tract and provides a higher and longer sustained serum level than penicillin V. Table 15-2 presents alternative regimens for endocarditis prophylaxis. Guidelines for antibiotic prophylaxis for patients with total joint replacements also have been recently updated. Those considered at risk for prosthetic joint replacement include immunocompromised or immunosuppressed patients, patients with insulin-dependent (i.e., type I) diabetes, patients who had joint replacement surgery less than 2 years previously, patients who have had previous prosthetic joint infections, malnourished patients, and patients with hemophilia. The recommended antibiotic regimen is 2 g of amoxicillin 1 hour before dental treatment. If the patient is unable to take oral medications, cefazolin (1 g) or ampicillin (2 g) should be given intramuscularly or intravenously 1 hour before the dental procedure. If the patient is allergic to penicillin, clindamycin (600 mg) should be given orally 1 hour before the dental treatment. Clindamycin (600 mg) may be given intravenously if the patient is unable to take oral medications. MICROBIAL SAMPLES FOR LABORATORY SUPPORT Table 15-2. Prophylactic Regimens for Dental Procedures SITUATION AGENT REGIMEN Standard general Amoxicillin Adults: 2 g

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prophylaxis

Patient unable to take Ampicillin oral medications

Patient allergic to penicillin

Clindamycin

Cephalexin or cefadroxil

Azithromycin or clarithromycin

Patient allergic to penicillin and unable to take oral medications

Clindamycin or Cefazolin

Children: 50 mg/kg Given orally 1 hour before procedure Adults: 2 g Children: 50 mg/kg Given intramuscularly (IM) or intravenously (IV) within 30 min before procedure Adults: 600 mg Children: 20 mg/kg Given orally 1 hour before procedure Adults: 2 g Children: 50 mg/kg Given orally 1 hour before procedure Adults: 500 mg Children: 15 mg/kg Given orally 1 hour before procedure Adults: 600 mg Children: 20 mg/kg Given IV within 30 min before procedure Adults: 1 g Children: 25 mg/kg Given IM or IV within 30 min before procedure

From Dajani et al: Prevention of bacterial endocarditis, J Am Med Assoc 277(22):1794, 1997. identification of the organisms or susceptibility tests may be valuable in some cases. For example, infective organisms that are better targeted by an antibiotic selected with laboratory support may be required for patients who are immunosuppressed after radiation or chemotherapy or who are at high risk of developing an infection (e.g., previous infective endocarditis). Samples may be collected either from an infected root canal or from a periradicular abscess or cellulitic area. To obtain an aseptic sample from a root canal, the clinician must isolate the tooth with a rubber dam. The surface of the tooth and surrounding field must be disinfected with sodium hypochlorite (NaOCl) or some other disinfectant. Access to the canal is created with sterile burs and instruments. If drainage is present, the sample may be

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collected with a sterile needle and syringe or sterile paper points. Air should be vented from the syringe and the aspirate placed in anaerobic transport medium provided by the laboratory. To sample a dry canal, the clinician should use a sterile syringe to place some sterile transport medium into the canal. A sterile instrument is used to suspend microbes in the medium (e.g., a sterile file is used to generate dentinal shavings), and the sample then is taken as described. A sample from a mucosal swelling is best acquired by needle aspiration through the disinfected mucosal surface to avoid contamination with "normal oral flora." Once profound anesthesia has been achieved, the patient is asked to rinse with chlorhexidine mouthwash and the surface is scrubbed with an iodophor swab. The surface then is penetrated with a sterile 16- to 20-gauge needle, the needle is moved into the area of swelling, and a sample of the exudate is aspirated. A new sterile needle is then placed on the syringe, and any air is vented before the aspirate is injected into an anaerobic transport medium recommended by the laboratory. After the specimen has been obtained, an incision for drainage is made, and blunt dissection with a periosteal elevator and curved hemostat is performed to provide complete drainage from the tissues.

THEORY OF FOCAL INFECTION-DJ VU?


In 1952 an editorial in the Journal of the American Medical Association stated: After exerting a tremendous influence on the practice of medicine for a generation, the theory of focal infection in the past 10 or 15 years has fallen in part into disfavor. This has been due partly to the following observations that seem to discredit it: (1) Many patients with diseases presumably caused by foci of infection have not been relieved of their symptoms by removal of the foci; (2) many patients with these same systemic diseases have no evident focus of infection; (3) foci of infection are, according to some statistical studies, as common in apparently healthy persons as in those with disease.

The origin of the focal infection theory dates to 1888, when Dr. W.D. Miller proposed that necrotic pulp could act as a center of infection, resulting in an alveolar abscess. In 1904 Dr. Frank Billings reported for the first time a positive correlation between oral disease and endocarditis and defined a focus of infection as a "circumscribed area of tissue infected with pathogenic organisms." In 1909 Dr. E.C. Rosenow, a student of Billings, reported that organisms in diseased organs could establish an infection in a distant organ. This "theory of elective localization" proposed that bacteria have a specific affinity for certain tissues and organs of the body. Rosenow went on to define focal infection as a "localized or generalized infection caused by the dissemination of bacteria or their toxic products from a distant focus of infection." As a result of these clinical and scientific studies, both the medical and dental

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professions concluded that no relationship existed between teeth with nonvital pulps or endodontically treated teeth and any of the so-called degenerative diseases. Although the focal infection theory resulted in needless extraction of millions of teeth, it definitely encouraged research that eventually led to the current scientific and biologic basis for root canal treatment.

ASSOCIATION OF ORAL AND SYSTEMIC DISEASE Practitioners are well aware of the relationship between bacteremias caused by various dental procedures and the resultant risk of infective endocarditis in patients with damaged heart valves as a result of congenital or rheumatic heart disease. It must be pointed out that a bacteremia can also result from normal chewing and tooth brushing. Infective endocarditis is a classic example of a distant secondary infection that is not related to the focal infection theory proposed in 1910. The bacteremia occurs at the time of the dental procedure, not from bacteria leaking from a nonvital tooth or from endodontically treated teeth. Although the focal infection theory as it relates to teeth with nonvital pulps and endodontically treated teeth has been disproved, growing evidence indicates that a relationship does indeed exist between oral and systemic disease. epidemiologic and case-based studies have shown a positive correlation between periodontitis and cardiovascular and cerebrovascular diseases and preterm low-birth-weight babies. In 1989 Mattila et al found a positive relationship between poor dental health and acute myocardial infarction. DeStefano et al reported in 1993 that subjects with periodontitis had a 25% increased risk of coronary heart disease compared to those with minimal periodontal disease. Hypothesizing that periodontal disease, which is a chronic Gram-negative infection, represents a previously unrecognized risk factor for atherosclerosis, investigators conducted a cohort study of periodontal and cardiovascular disease. Subjects with bone loss greater than 20% had approximately twice the incidence of fatal coronary heart disease; for every 20% increase in mean bone loss, the incidence of total coronary heart disease increased 40%. A case control study in which all known risk factors and covariates for preterm low birth weight were controlled found that periodontal disease resulted in a sixfold increase in the risk for preterm low birth weight. Therefore, although a review of the scientific literature finds that most studies reported a positive correlation between oral disease and cardiovascular

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disease, stroke, and preterm low birth weight, several studies did not. Many questions remain regarding the exact role of oral bacteria in the onset and progression of systemic disorders. A cause-and-effect relationship between periodontal and systemic disease must be studied, as must the relevance of endodontic infections to systemic disease. Nevertheless, the point must be emphasized: No scientific evidence exists that bacteria remaining in the dentinal tubules after properly performed endodontic treatment are a source of bacteria for chronic systemic diseases. SUMMARY It is important that clinicians understand the close relationship of microorganisms with endodontic disease to develop an effective treatment rationale. This chapter describes the microbes commonly associated with endodontic disease and their relationship to pulpal and periradicular infections. The clinical management of endodontic infections is described along with the appropriate use of adjunctive antibiotics. In addition, the antibiotics appropriate for the treatment of medically compromised patients are described, and information is presented on the association of oral, and systemic disease.

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