https://orcid.org/0000-0001-5017-1340
Figures
Abstract
Fungal infections are common on oral mucosae, but their role in other oral sites is ill defined. Over the last few decades, numerous studies have reported the presence of fungi, particularly Candida species in endodontic infections, albeit in relatively small numbers in comparison to its predominant anaerobic bacteriome. Here, we review the fungal biome of primary and secondary endodontic infections, with particular reference to the prevalence and behavior of Candida species. Meta-analysis of the available data from a total of 39 studies fitting the inclusion criteria, indicate the overall weighted mean prevalence (WMP) of fungal species in endodontic infections to be 9.11% (from a cumulative total of 2003 samples), with 9.0% in primary (n = 1341), and 9.3% in secondary infections (n = 662). Nevertheless, WMP for fungi in primary and secondary infections which were 6.3% and 7.5% for culture-based studies, increased to 12.5% and 16.0% in molecular studies, respectively. The most prevalent fungal species was Candida spp. The high heterogeneity in the reported fungal prevalence suggests the need for standardized sampling, and speciation methods. The advent of the new molecular biological analytical platforms, such as the next generation sequencing (NGS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), that enables identification and quantitation of a broad spectrum of hitherto unknown organisms in endodontic infections should radically alter our understanding of the endodontic mycobiome in the future. Candida spp. appear to be co-pathogens with bacteria in approximately one in ten patients with endodontic infections. Hence, clinicians should comprehend the importance and the role of fungi in endodontic infections and be cognizant of the need to eradicate both bacteria and fungi for successful therapy.
Citation: Alberti A, Corbella S, Taschieri S, Francetti L, Fakhruddin KS, Samaranayake LP (2021) Fungal species in endodontic infections: A systematic review and meta-analysis. PLoS ONE 16(7): e0255003. https://doi.org/10.1371/journal.pone.0255003
Editor: Supram Hosuru Subramanya, Manipal College of Medical Sciences, NEPAL
Received: March 22, 2021; Accepted: July 7, 2021; Published: July 22, 2021
Copyright: © 2021 Alberti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Fungi are common opportunistic pathogens, and they comprise a substantial proportion of the human microbiome [1]. The fungal human microbiome, termed the mycobiome has numerous constituents, but the predominant of which belongs to the genus Candida [2]. Of over 200 species of human pathogenic and saprophytic, Candida species the following, which are of major medical importance occupy the top positions in the hierarchy of the Candida biome: C. albicans, C. glabrata, C. kefyr, C. krusei, C. parapsilosis and C. tropicalis. Of these, C. albicans is, by far, the commonest intra oral species, in 30–45% healthy adults, and the main agent of oral candidal infections [3]. Two reviews of the microbiology of endodontic infections reported C. albicans as the most common isolate from the endodontic mycobiome [4,5].
The transition of Candida from a harmless oral commensal to an opportunist pathogen depends on a number of predisposing conditions, including immune functionality, endocrine disorders, ill-fitting dentures, poor oral hygiene, use of broad-spectrum antibiotics, corticosteroids, immunosuppressive agents, and drugs that may induce neutropenia and xerostomia [3]. Candida species, in general possess an armamentarium of virulence attributes which, acting in concert may cause candidiasis in vulnerable individuals when an opportunity arises [1,6,7]. These include i) adhesion and biofilm formation on biotic and abiotic host surfaces, mediated by surface molecules [6]; ii) production of hydrolytic enzymes, proteinases, phospholipases, and hemolysins which degrade extracellular matrix proteins of the host tissues [6,7]; iii) thigmotropism; iv) phenotypic switching and consequent environmental adaptability; v) evasion of host immune system, through degradation of IgG1, IgA1, and IgA2 [6,7] and suppressing polymorphonuclear neutrophil functions; and vi) immunomodulation through stimulation of proinflammatory cytokine synthesis and activation of the complement cascade [6].
Candida spp. have a predilection to reside in a number of specific oral niches. The dorsum of the tongue is considered its primary oral habitat [1] although the fitting surface of acrylic denture surface is the major reservoir of the yeasts in denture wearers. Nevertheless, it can habituate other oral mucosal sites, as well as supra- and sub-gingival plaque biofilms [8,9], periodontal pockets [10], carious lesions and infected root canals (syn. endodontium) [6].
The role of Candida spp. in carious and endodontic infections is ill defined. However, there is a growing body of data to implicate that the Candida biome play a significant role in the pathogenesis of dental caries and the resultant endodontic sequelae [11–13], as discussed below.
In a seminal recent review, Pereira et al. [14] have elegantly argued the plausible basis of the cariogenic potency of Candida. In clinical terms, the isolation of Candida spp. from enamel, dentine and root caries [15], the correlation between high salivary Candida carriage and the severity of caries [16,17], and its high prevalence in early childhood caries [18] demonstrate that Candida spp. may have a significant impact on the carious process.
Further examination of the pathogenic attributes of Candida spp. that define it as a candidate cariogen is salutary. For instance, in vitro studies reveal that C. albicans is able to firmly adhere to normal or EDTA/NaOCl-treated enamel, dentine, cementum surfaces [19,20], and it has a very high affinity for hydroxyapatite [19,21,22]. The presence of the smear layer increases the adhesion of C. albicans to human dentin [23,24], probably due to the availability of exposed dentinal collagen and calcium ions. Indeed, Candida is able to bind to collagen types I and IV [6,25–27], and it has a specific affinity for dentinal collagen. Furthermore, calcium ions modify Candida morphogenesis and its capacity to adhere to extracellular matrix proteins [23,24,28]. Due possibly to its thigmotrophic properties, C. albicans has the unique ability to penetrate deep into exposed dentinal tubules in carious lesions. For these reasons, some have called the yeast a “dentinophilic” microorganism [29]. In terms of community living within cariogenic niches, many Candida spp. have the potential to co-aggregate with cariogenic bacteria [6,14]. Interestingly, Falsetta et al. [30] reported that C. albicans induces virulence gene expression in S. mutans, that in turn may facilitate bacterial-yeast aggregation.
Many Candida spp. can rapidly metabolize dietary carbohydrates such as glucose, sucrose, fructose, and even polyols (e.g. xylitol) [14], leading to the formation of acidic end-products such as short-chain carboxylic acids, lactate and acetates that demineralize enamel. The acidification is also due to the activation of H+-ATPase-dependent sugar uptake system of C. albicans plasma membrane, and to the generation of carbon dioxide from glucose metabolism [31]. Such acid production in a localized, focal biofilm niche causes a rapid drop of the pH to 5.5 or below leading to enamel and dentine demineralization [14,19,32]. The reduction of pH also leads to the activation of acid proteases, collagenases, and phospholipases of Candida spp., that assist degradation of the dentinal collagen [25,26,33,34]. In conclusion, Candida could have a deleterious cariogenic effect on dental hard tissues through independent, dichotomous mechanisms: first, by dissolving the inorganic matrix of either enamel or dentine with its metabolic acids, and second, by disassembling the organic matrix by its abundant collagenolytic enzymes.
There are also a number of studies that points to a critical role of Candida in the pathogenesis of endodontic infections. The yeast, being a microaerophilic eukaryote possesses the metabolic armory necessary to survive within the harsh and barren ecosystem of the root canal. Several workers have shown that C. albicans could use dentin itself as a source of nutrition in vitro, in the absence of other extraneous food supplements, and colonize the canal walls as well as the dentinal tubules [29,35,36]. In one experiment, the penetration of C. albicans into dentinal tubules in vivo was shown to be facilitated by the presence of a smear layer, produced by instrumentation [37]. The sheltered life of the yeast within the tubules, out of reach of instruments, and disinfectant irrigants, is also likely to perpetuate chronic endodontic infections [6,38].
With regard to the localization of fungi within the root canal system, Nair et al. [39] elegantly demonstrated through ultrastructural imaging, the presence of yeast-like organisms in root canals, and at the apical foramen. Persoon et al. [40] also observed dense masses of yeast cells in root canals, while dentinal tubules were filled with hyphae. Siqueira et al. [41] investigated the patterns of microbial colonization in primary root canal infections through scanning electron microscopy, and observed single or budding yeast cells in root canal systems.
Nevertheless, since then, there has been an explosive advancement in microbial identification techniques, and new data are available on the fungi in this oral eco-niche. Hence, the aim of this systematic review and meta-analysis was to review the recent literature on the prevalence of fungal species in primary and secondary endodontic infections.
Materials and methods
Data sources
Two investigators (AA and SC) performed an electronic search of MEDLINE/PubMed, SCOPUS, ISI Web of Science, and Cochrane Central without any language restrictions published since 2000. Grey literature was searched for pertinent papers by using OpenGrey and Greylist. A manual search was performed of all the issue published since 2000 of Journal of Endodontics, International Endodontic Journal and Oral Surgery, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology. Moreover, reference lists of all included papers were screened for articles that need to be evaluated for inclusion in the study. The final electronic search was performed on 10th March 2021. Our investigation was focused on relatively recent literature, in view of the fact that older, conventional microbiological analytical techniques, mainly depending on phenotypic analyses, have undergone a radical revolution with the advent of molecular biological screening techniques, such as next generation sequencing (NGS) platforms.
The specific review question formulated was as follows: What is the prevalence of fungal species in primary and secondary endodontic infection in human permanent teeth?.
Search terms
A specific search string was formulated for each of the databases, which included each of the following group of search terms, combined by means of the Boolean operator AND:
- (Dental pulp disease?) OR (apical periodont*) OR (periapical periodont*) OR (apical disease?) OR (periapical disease?) OR (periapical lesion?) OR (apical lesion?) OR pulpitis OR (dental pulp necrosis) OR (tooth necrosis) OR (endodontic lesion?) OR (endodontic pathosis).
- Fung* OR mycet* OR mycos* OR candid* OR yeast? OR microb*
- Exp dental pulp diseases OR exp periapical diseases OR exp periapical periodontitis OR Exp fungi
Study selection
Inclusion criteria.
Human endodontic studies; studies on the detection of fungal species within infected root canal of either carious teeth or after failed endodontic treatment; studies clearly relating method of preparation of the sampling site and sample collection; clear description of the methodology employed for fungal detection; use of standardized methods for sample collection, transportation, and analysis (e.g., isolation with a rubber dam, use of sterile paper point; studies on permanent teeth with complete root formation.
Electronic data-search and assessment
To answer the review question, a four-step approach of evidence-based analysis was employed. According to the pre-set inclusion and exclusion criteria, the title and abstract were independently screened by two authors (AA and SC) at stage-one; full texts of the included records were assessed for eligibility by the same authors (AA and SC). Disagreements in article selection processes were solved by consulting a third reviewer (LS). At stage-two of the data extraction, the author (AA) meticulously screened the included full texts and extract information on sample size, primary or secondary infection after endodontic treatment. Each study’s characteristics were recorded employing the Cochrane pattern determining the study design, setting, methods sample collection, culture medium, yeast species recovered. Additionally, the microbiological evaluation and outcomes about the prevalence of fungal species were recorded in a spreadsheet. The authors were contacted via emails if insufficient information were available from the included studies.
Quality of the evaluated studies.
At the third stage of the review, a critical appraisal of the included studies was performed adopting a modification of the checklist proposed by Hoy and colleagues [42] using the following criteria: i) was the target population close representation of the national population in relation to relevant variables? ii) was the sampling frame a true or close representation of the target population? iii) were data collected directly from the subjects? iv) was the study instrument that measured the parameter of interest shown to have reliability and validity? v) was the same mode of data collection used for all subjects? Based on the above criteria a score was assigned for each study. A score of 0 is low risk bias, while (1–2) is considered as moderate risk and a score between (3–5) reflects high risk of bias, respectively.
Summary measures, synthesis of results, and other analysis
The fourth and final stage involved the analysis of the results, performed by an author (SC) by using R (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/).
Data on the prevalence of fungal species were extrapolated from the studies and pooled together by calculating the weighted mean prevalence (based on the number of samples in each study). Separate analyses were performed for i) primary and the secondary endodontic treatment; ii) methods used for evaluating the presence of fungal species (molecular, cultural, or both), iii) the collection method (e.g., paper points and endodontic hand files), and iv) studies with periapical lesions in systemically healthy subjects. The meta-analysis was performed using the DerSimonian-Laird random-effects method (assuming heterogeneity among studies), calculating pooled proportion.
Cochran’s test served to measure the results’ consistency, with a significant level set at P <0.1. The heterogeneity of the outcomes was also evaluated as follows: <40% the heterogeneity was considered negligible, 41% to 60% moderately heterogeneous, if 61% to 90% substantially heterogeneous [43].
Results
The initial search identified 5,255 articles. Among them, 55 were duplicates and hence excluded. Of the remaining 5,200 papers, 5,110 were excluded after perusal of the titles and abstracts as they did not fit the inclusion criteria. Further screening for full texts resulted in the exclusion of 52 articles, as the outcomes were incongruent with the study objectives. Finally, a total of 39 studies using culture and molecular fungal assessment methods were included in the current review [44–82]. Twenty-three included studies used the culture method for fungal evaluation. Among them, eight studies examined samples from primary endodontic infection, while ten, assessed the presence of fungal species in teeth with secondary endodontic infection. Another five studies evaluated the fungal presence in either primary or secondary endodontic infections. Of nineteen molecular studies reviewed, 12 studies examined the fungal presence in primary endodontic infection, while six appertained to secondary infection, and a single study in either a primary or a secondary infection. Fig 1 represents the articles selection process. Tables 1–6 present the characteristics of the included studies which investigated the yeast colonization of root canals in primary and secondary pulp infections through cultural methods and molecular analytical techniques, respectively. The prevalence of fungi in primary endodontic infections was investigated in 25 studies, with a total of 1341 analyzed samples and a weighted mean prevalence (WMP) of 9.0% (CI 95%: 6.0–12.0) (Fig 2). We found a significant heterogeneity among papers with regard to the reported values, as the prevalence of fungal species varied between 0 and 46.8% [78].
As for studies using cultural methods, a total of 535 specimens was analyzed: the prevalence of fungi ranged from 0 [54,72] to 41.67% [79] and the WMP was 6.3% (CI 95%: 2.9–9.8) (Fig 3). On the other hand, when molecular biological methods were used, the prevalence ranged from 0.00 to 46.88% [78], yielding a WMP of 12.5% (CI 95%: 7–18) in a total of 779 samples (Fig 3). When both techniques were combined, then the WMP was 1.8% [72] (Fig 3).
The studies that reported higher yeast prevalence [78,79] were based only on a small sample size (36 and 32 samples, respectively), while three of the studies with the highest number of clinical samples were based on PCR techniques and reported a prevalence of 19.66% [71], 22.62% [64] and 28.33% [80].
As for persistent infections, the prevalence of fungi was reported to range between 0.00 and 42.86% [77], and the calculated WMP was 9.3% (CI 95%: 5.8–12.8) from a total of 662 samples (Fig 2). Cumulative culture data indicate that 434 specimens yielded 44 positive samples. The resultant WMP of yeasts was 7.5% (CI 95%: 3.7–11.2). In one case, a higher prevalence rate (42.86%) was found in a cohort of only six positive specimens, that may have led to high level of skewing of the results [77]. When PCR was used, the calculated WMP was 16.0% (CI 95%: 6.7–25.4) (Fig 4). In two studies [69,75], both techniques were used, resulting in a WMP of 5.5% (CI 95%: -3.2–14.3) (Fig 4).
Considering all endodontic infections evaluated, the mean prevalence of fungi was found to be 9.11% (CI 95%: 6.8–11.4) in a total of 2003 specimens. The foregoing clearly implies that fungi contribute to up to one in ten to one in eleven root canal infections and re-infections. Interestingly, no differences in WMP were found among the detecting methods for each lesion type (either primary or secondary).
A total of seven workers [45,50,55,62,80–82] used both paper points and hand files for sample collection. Only one of them [45] used cultural methods, thus the meta-analysis could not be performed, while six of them [50,55,62,80–82] used PCR, and the resulting WMP was 15.6% (CI 95%: 5.8–25.4) (Fig 5). Thirteen studies only included teeth presenting periapical lesion, with a WMP of 5.4% (CI 95%: 2.2–8.6) for cultural studies [45,47,48,51,52,57,58,65,66,72] (Fig 6) and 18.7% (CI 95%: 2.9–34.5) for molecular studies [55,64,72,78] (Fig 7). Studies including only systemically healthy subjects were also separately analyzed, and the calculated WMP for the latter group was 6.6% (CI 95%: 3.8% - 9.4%) for 15 cultural studies, and 16.6% (CI 95%: 5.3–27.8) for six molecular studies (Figs 8 and 9).
The majority of the studies evaluated were deemed to be at low risk of bias, while 10 studies were considered as moderate risk. In most cases, the risk was associated to an incomplete description of the target population, and the lack of randomization of the samples, which could undermine the external validity of the results. For example, only mandibular molars [75,78], permanent teeth from pediatric patients [62], symptomatic [73] or asymptomatic teeth [48,58,66–69,72,75], or a limited age range were included [74], and in several studies that may have led to inherent bias. On the other hand, there were inadequate number of studies in each of the foregoing subgroups to conduct metanalyses.
In several other cases, the augmented risk of bias was increased due to poor or total absence of the description of the tooth and the sample site preparative procedures prior to the microbiological sampling [47,53,54,74,79]. On the contrary, some studies provided very precise descriptors of the protocols for sample selection (e.g. collection from the largest root canal associated with the periapical lesion) including the quality of disinfection of the sampling site, and single or multiple control specimens [45,46,48,57–59,64,67–69,71,72,75,76,81,82] all of which affect the microbiological outcomes. It must also be noted that all the included studies used rubber dam isolation procedures of the tooth before sampling procedures.
Discussion
Our meta-analysis of the literature comparing the fungal biome of primary and secondary endodontic infections, comprised selectively curated 39 studies, performed over a period of approximately two decades. These indicate the presence of fungal infection in approximately one in ten endodontic infections, with similar prevalence rates in both the primary (9.0%) and secondary infections (9.3%). A great heterogeneity was observed in the analyzed data, which represents a limitation of the present study. There are a number of reasons for the heterogeneity of the data that were noted, and they are essentially dichotomous in nature and falls into two broad categories, the health and demographic characteristics of the studied subjects, and the microbiological sampling and evaluation technology.
The vast majority of the cases included in the review were from systemically healthy and immunocompetent adults, not undergoing antibiotic or antifungal therapy over 1–6 months prior to sampling. However, a minority of studies were in children [62], and some were in human immunodeficiency virus (HIV) infected [70], and diabetic subjects [80]. These accounted for a limited number of cases (146 samples, respectively from 66 children, 20 HIV positive and 60 diabetic patients). Interestingly, the diabetic patients presented with a high prevalence of C. albicans (38.3%) in the infected root canals [80].
The microbiological sampling and evaluation technology appear to have played a critical role in the disparate outcomes reported here irrespective of whether they were based on traditional microbial culture technology, or molecular biological studies. Culture or molecular technology outcomes are essentially a direct reflection of the sample quality [83], which can be significantly impacted by the poor isolation of the sampling site, inadequate asepsis and disinfection of the entry portal (of the sampling material), poor access cavity design, missed or obtuse canals, inadequate instrumentation and debridement to remove superficial debris and/or saliva contamination, and prior contamination of the root canal system due to seepage of the contaminant oral flora through poorly sealed temporary or permanent restorations (in the case of secondary infections).
As regards the sample collection protocols, multiple differences were found among studies. Paper point absorption method was the most commonly used, and proved to be the more sensitive according to one group [45], although endodontic instruments such as K-type or Headstrom files were used for sample collection by a large number of workers [45,50,55,61,62,70,80–82]. There were inherent deficiencies in some studies as chemicals, that may affect fungal viability, were used during the mechanical instrumentation by the researchers, prior to sample collection [80]. As for persistent chronic infections, no chemical solvents were used by any of the investigators, to remove the root filling except in one study by Egan et al. [49].
Varying protocols for field decontamination were used prior to the endodontic access by different workers, including the use of iodine, hydrogen peroxide, chlorhexidine gluconate, sodium hypochlorite, or isopropyl alcohol of varying dilutions [45,49,50,55,57,61,63,64,67–69,71–73,75,78,81,82]. It must be noted that liquid disinfectants may flow into the pulp chamber and interfere with the sampling if applied after endodontic access or when the endodontic space communicates with the oral cavity. Sodium thiosulfate was also used in some studies to inactivate iodine, so as not to impact the microbiological sampling quality. Some of the protocols also employed sterility checks using a control samples to ascertain the integrity of the sampling technique [45,46,48,57–59,64,67–69,71,72,75,76,81,82].
Another noteworthy point is the microbiological culture methods used by different investigators which may lead to discrepant results. Due to the quantitatively low numbers of fungi populating the infected root canal systems their growth may take upto 72 hr to be discernible as colony forming units (CFU) unless appropriate culture media are used and observed for at least three days. If the laboratory workers do not use fungal culture media (such as Sabourauds agar or chromogenic agar) and/or evaluates the culture results only upto 24 hr, then there is a high likelihood that the yeast prevalence will be under reported. However, this deficiency could be overcome through molecular mycological analyses using fungal specific universal primers, when the total culturable as well as unculturable fungal flora could be detected with confidence, irrespective of their population size within the endodontic ecosystem.
Another limitation of the study is that we were not able to perform a patient-level analysis of the prevalence of fungi in endodontic infections. Although the majority of the included studies collected and analyzed only one sample per patient, some of them included more samples from the same subjects [49,57,60,64,71,76,79], while in some other papers this was not specified [44,46,50–52,56,59,61,63,65,73].
Microorganisms may still persist within the root canal system even after adequate debridement and disinfection and related clinical procedures. This is due to the complex anatomy and the architecture of the root canal system that may include the presence of accessory canals, anatomical lacunae, and delta spaces. A number of workers have compared the prevalence of fungi in primary endodontic infections before and after endodontic therapy, and some have noted the increased prevalence of yeasts and other fungal species, post treatment, even though, in a few cases, they were undetectable prior to treatment [46,51,58]. The same findings had been already reported in previous studies [84,85]. This implies that fungi have the potential to often survive a harsh, nutritionally depleted and chemically infused ecosystem consequential to endodontic treatment procedures [86] and may gain access into the root canal system during or after the endodontic treatment.
Various studies have analyzed the inter kingdom co-habitation of yeasts and bacteria in the infected endodontic ecosystems. Although C. albicans is typically isolated from persistent endodontic infections in mixed cultures of cohabitant oral bacteria, at least four groups have reported identification of pure cultures of Candida from root canal infections [36,46,87,88]. Thus, Hancock et al. in a very early report, over two decades ago, described the mono-infection of root canals with C. albicans [45]. Also, in one of the largest studies to date, Waltimo and coworkers reported isolation of fungi in pure cultures from six of 967 endodontic samples, and yeast/bacterial mixed cultures in another 41 cases [86].
Another major reason for the heterogeneity of microbiological results we noted, could be the shift from cultural studies to molecular biological studies for microbiome evaluation, over the last few decades. Together with the increased sophistication of the molecular technologies there has been a parallel increase in the knowledge of the oral microbiome, leading to the elucidation of a hitherto unknown universe of unculturable flora. Accordingly, the cumulative data from our review clearly indicate that PCR analytical techniques yield higher fungal prevalence values as compared with the culture methods. This is clearly seen in WMP for fungi in primary and secondary infections which were 6.3% and 7.5% for pure culture-based studies, that increased to 12.5% and 16.0% in PCR studies, respectively. Adding to this confusion, some workers used both cultural and molecular techniques for mycological analyses, and additional positive cases were detected through PCR analyses [69,72,75]. This was also the case in a recent study by Al-Sakati et al. [88], who compared culture-dependent and culture-independent methods to detect bacteria and fungi in reinfected root-filled teeth, and found a higher detection rate of fungi using the latter techniques.
Apart from the low population density of fungi in infected root canal systems, there may be other reasons for heterogeneous results even within molecular mycological analyses. These include the use of dissimilar primers by different investigators, with varying degrees of specificity. Al-Sakati et al. [88] noted, for instance, an increased rate of fungal detection by improving the specificity of the primers used. One disadvantage of the use of multiple specific primers instead of a single universal primer is the increased costs and time associated with the former approach. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) has been proposed for fungal identification [89]. The absence of a comprehensive and well-curated spectral database of fungi, is the major limitation of this technique, although they are becoming increasingly robust and complete as time progresses with input from ongoing studies. MALDI-ToF MS analyses being inexpensive, easy, fast and accurate, and the fact that it can be applied to detect a comprehensive array of microorganism implies that the method would be very useful for future workers investigating the endodontic mycobiome. We reviewed, two studies which compared MALDI-ToF MS analysis and conventional cultural tests, but these workers failed to detect any fungal isolates from the endodontium [69,75].
Another exciting development in the quantitative and qualitative microbiological analyses is the advent of the Next Generation Sequencing (NGS) platforms. These, based on various platforms such as Illumina and Ion-torrent, as well as novel inexpensive, miniaturized platforms such as Minion technology, hold much promise in the demystification of the fungal flora of infected root canal systems.
Conclusions
The role of fungi in the pathogenesis of endodontic infections is unclear, as yet. The significant heterogeneity of results in the reviewed studies reflect the variety of the techniques used for sample collection, culture and identification. On the other hand, novel molecular techniques such as MALDI-TOF and NGS analyses should elucidate the true mycobiome as the new technology could redefine both the culturable and unculturable mycobiota in endodontic infections. Finally, as almost one in ten endodontic infections are associated with fungal infestation, clinicians must pay heed the need to eradicate both bacteria and fungi from infected root canal systems during treatment procedures.
References
- 1.
Samaranayake LP. Fungi of relevance to dentistry. In: Samaranayake LP, editor. Essential Microbiology for Dentistry. 5th ed. Edinburgh: Elsevier; 2018. p. 187–92.
- 2. Seed PC. The human mycobiome. Cold Spring Harb Perspect Med. 2014;5(5):a019810. pmid:25384764
- 3.
Samaranayake LP, MacFarlane T. Oral Candidosis. Bristol: Wright; 1990.
- 4. Mergoni G, Percudani D, Lodi G, Bertani P, Manfredi M. Prevalence of Candida Species in Endodontic Infections: Systematic Review and Meta-analysis. J Endod. 2018;44(11):1616–25 e9. pmid:30241680
- 5. Persoon IF, Crielaard W, Ozok AR. Prevalence and nature of fungi in root canal infections: a systematic review and meta-analysis. Int Endod J. 2017;50(11):1055–66. pmid:27987307
- 6. Siqueira JF Jr., Sen BH. Fungi in endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004;97(5):632–41. pmid:15153878
- 7. McCullough MJ, Ross BC, Reade PC. Candida albicans: a review of its history, taxonomy, epidemiology, virulence attributes, and methods of strain differentiation. Int J Oral Maxillofac Surg. 1996;25(2):136–44. pmid:8727588
- 8. Arendorf TM, Walker DM. The prevalence and intra-oral distribution of Candida albicans in man. Arch Oral Biol. 1980;25(1):1–10. pmid:6996654
- 9. Slots J, Rams TE, Listgarten MA. Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis. Oral Microbiol Immunol. 1988;3(2):47–52. pmid:3268751
- 10. Reynaud AH, Nygaard-Ostby B, Boygard GK, Eribe ER, Olsen I, Gjermo P. Yeasts in periodontal pockets. J Clin Periodontol. 2001;28(9):860–4. pmid:11493356
- 11. Simon-Soro A, Mira A. Solving the etiology of dental caries. Trends Microbiol. 2015;23(2):76–82. pmid:25435135
- 12. Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, et al. Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol. 2008;46(4):1407–17. pmid:18216213
- 13. Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, et al. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol. 2002;40(3):1001–9. pmid:11880430
- 14. Pereira D, Seneviratne CJ, Koga-Ito CY, Samaranayake LP. Is the oral fungal pathogen Candida albicans a cariogen? Oral Dis. 2018;24(4):518–26. pmid:28513096
- 15. Zaremba ML, Stokowska W, Klimiuk A, Daniluk T, Rozkiewicz D, Cylwik-Rokicka D, et al. Microorganisms in root carious lesions in adults. Adv Med Sci. 2006;51 Suppl 1:237–40. pmid:17460839
- 16. Lozano Moraga CP, Rodriguez Martinez GA, Lefimil Puente CA, Morales Bozo IC, Urzua Orellana BR. Prevalence of Candida albicans and carriage of Candida non-albicans in the saliva of preschool children, according to their caries status. Acta Odontol Scand. 2017;75(1):30–5. pmid:27796162
- 17. De-la-Torre J, Marichalar-Mendia X, Varona-Barquin A, Marcos-Arias C, Eraso E, Aguirre-Urizar JM, et al. Caries and Candida colonisation in adult patients in Basque Country (Spain). Mycoses. 2016;59(4):234–40. pmid:26756815
- 18. de Carvalho FG, Silva DS, Hebling J, Spolidorio LC, Spolidorio DM. Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries. Arch Oral Biol. 2006;51(11):1024–8. pmid:16890907
- 19. Nikawa H, Yamashiro H, Makihira S, Nishimura M, Egusa H, Furukawa M, et al. In vitro cariogenic potential of Candida albicans. Mycoses. 2003;46(11–12):471–8. pmid:14641620
- 20. Sen BH, Safavi KE, Spangberg LS. Colonization of Candida albicans on cleaned human dental hard tissues. Arch Oral Biol. 1997;42(7):513–20. pmid:9296271
- 21. Cannon RD, Nand AK, Jenkinson HF. Adherence of Candida albicans to human salivary components adsorbed to hydroxylapatite. Microbiology. 1995;141 (Pt 1):213–9. pmid:7894715
- 22. Nikawa H, Hamada T, Yamashiro H, Murata H, Subiwahjudi A. The effect of saliva or serum on Streptococcus mutans and Candida albicans colonization of hydroxylapatite beads. J Dent. 1998;26(1):31–7. pmid:9479923
- 23. Klotz SA, Rutten MJ, Smith RL, Babcock SR, Cunningham MD. Adherence of Candida albicans to immobilized extracellular matrix proteins is mediated by calcium-dependent surface glycoproteins. Microb Pathog. 1993;14(2):133–47. pmid:8502162
- 24. Turk BT, Ates M, Sen BH. The effect of treatment of radicular dentin on colonization patterns of C. albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;106(3):457–62. pmid:18707654
- 25. Kaminishi H, Hagihara Y, Hayashi S, Cho T. Isolation and characteristics of collagenolytic enzyme produced by Candida albicans. Infect Immun. 1986;53(2):312–6. pmid:3525409
- 26. Hagihara Y, Kaminishi H, Cho T, Tanaka M, Kaita H. Degradation of human dentine collagen by an enzyme produced by the yeast Candida albicans. Arch Oral Biol. 1988;33(8):617–9. pmid:3075886
- 27. Makihira S, Nikawa H, Tamagami M, Hamada T, Nishimura H, Ishida K, et al. Bacterial and Candida adhesion to intact and denatured collagen in vitro. Mycoses. 2002;45(9–10):389–92. pmid:12421287
- 28. Holmes AR, Cannon RD, Shepherd MG. Effect of calcium ion uptake on Candida albicans morphology. FEMS Microbiol Lett. 1991;61(2–3):187–93. pmid:2037228
- 29. Sen BH, Safavi KE, Spangberg LS. Growth patterns of Candida albicans in relation to radicular dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1997;84(1):68–73. pmid:9247954
- 30. Falsetta ML, Klein MI, Colonne PM, Scott-Anne K, Gregoire S, Pai CH, et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect Immun. 2014;82(5):1968–81. pmid:24566629
- 31. Klinke T, Kneist S, de Soet JJ, Kuhlisch E, Mauersberger S, Forster A, et al. Acid production by oral strains of Candida albicans and lactobacilli. Caries Res. 2009;43(2):83–91. pmid:19246906
- 32. Samaranayake LP, Hughes A, Weetman DA, MacFarlane TW. Growth and acid production of Candida species in human saliva supplemented with glucose. J Oral Pathol. 1986;15(5):251–4. pmid:3091791
- 33. Samaranayake L.P. MTW. On the role of dietary carbohydrates in the pathogenesis of oral candidosis. FEMS Microbiol Lett. 1985;527:1–5.
- 34. Nishimura M, Nikawa H, Yamashiro H, Nishimura H, Hamada T, Embery G. Cell-associated collagenolytic activity by Candida albicans. Mycopathologia. 2002;153(3):125–8. pmid:11998872
- 35. Waltimo TM, Orstavik D, Siren EK, Haapasalo MP. In vitro yeast infection of human dentin. J Endod. 2000;26(4):207–9. pmid:11199719
- 36. Siqueira JF Jr., Rocas IN, Lopes HP, Elias CN, de Uzeda M. Fungal infection of the radicular dentin. J Endod. 2002;28(11):770–3. pmid:12470022
- 37. Kinirons MJ. Candidal invasion of dentine complicating hypodontia. Br Dent J. 1983;154(12):400–1. pmid:6576802
- 38. Waltimo TMT, Haapasalo M, Zehnder M, Meyer J. Clinical aspects related to endodontic yeast infections. Endod Top. 2004;9(1):66–78.
- 39. Nair PN, Sjogren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study. J Endod. 1990;16(12):580–8. pmid:2094761
- 40. Persoon IF, Buijs MJ, Ozok AR, Crielaard W, Krom BP, Zaura E, et al. The mycobiome of root canal infections is correlated to the bacteriome. Clin Oral Investig. 2017;21(5):1871–81. pmid:27771826
- 41. Siqueira JF Jr., Rocas IN, Lopes HP. Patterns of microbial colonization in primary root canal infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002;93(2):174–8. pmid:11862207
- 42.
Hoy D, Brooks P, Woolf A, Blyth F, March L, Bain C, et al. Assessing risk of bias in prevalence studies: modification of an existing tool and evidence of interrater agreement. J Clin Epidemiol. 2012;65(9):934–9. https://doi.org/10.1016/j.jclinepi.2011.11.014 pmid:22742910
- 43.
Deeks JJ, Higgins JPT, Altman DG. Analysing data and undertaking meta-analyses. In: Higgins JPT, Thomas J, Chandler , Cumpston M, Li T, Page MJ, et al., editors. Cochrane Handbook for Systematic Reviews of Interventions. 2nd ed. Chichester: John Wiley & Sons; 2019.
- 44. Baumgartner JC, Watts CM, Xia T. Occurrence of Candida albicans in infections of endodontic origin. J Endod. 2000;26(12):695–8. pmid:11471635
- 45. Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;91(5):579–86. pmid:11346739
- 46. Lana MA, Ribeiro-Sobrinho AP, Stehling R, Garcia GD, Silva BK, Hamdan JS, et al. Microorganisms isolated from root canals presenting necrotic pulp and their drug susceptibility in vitro. Oral Microbiol Immunol. 2001;16(2):100–5. pmid:11240863
- 47. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J. 2001;34(6):429–34. pmid:11556508
- 48. Cheung GS, Ho MW. Microbial flora of root canal-treated teeth associated with asymptomatic periapical radiolucent lesions. Oral Microbiol Immunol. 2001;16(6):332–7. pmid:11737655
- 49. Egan MW, Spratt DA, Ng YL, Lam JM, Moles DR, Gulabivala K. Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis. Int Endod J. 2002;35(4):321–9. pmid:12059932
- 50. Siqueira JF, Rocas IN, Moraes SR, Santos KR. Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections. Int Endod J. 2002;35(4):345–51. pmid:12059935
- 51. Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J. 2003;36(1):1–11. pmid:12656508
- 52. Pinheiro ET, Gomes BP, Ferraz CC, Teixeira FB, Zaia AA, Souza Filho FJ. Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. Oral Microbiol Immunol. 2003;18(2):100–3. pmid:12654099
- 53. Tsang PCS CF, Samaranayake LP. Detection of Treponema, Enterococcus, Streptococcus, and Candida species in root canal infections in Southern Chinese. J Dent Res. 2003;82(Sp Iss B):147–73.
- 54. Gomes BP, Pinheiro ET, Gade-Neto CR, Sousa EL, Ferraz CC, Zaia AA, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol. 2004;19(2):71–6. pmid:14871344
- 55. Siqueira JF Jr., Rocas IN. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004;97(1):85–94. pmid:14716262
- 56. Chakraborty P, Chattopadhyay UK. A study on the polymicrobial etiology of root canal infections in anterior non-vital teeth in a government hospital in Kolkata, India. Eur Rev Med Pharmacol Sci. 2005;9(2):113–6. pmid:15945500
- 57. Chu FC, Tsang CS, Chow TW, Samaranayake LP. Identification of cultivable microorganisms from primary endodontic infections with exposed and unexposed pulp space. J Endod. 2005;31(6):424–9. pmid:15917680
- 58. Ferrari PH, Cai S, Bombana AC. Effect of endodontic procedures on enterococci, enteric bacteria and yeasts in primary endodontic infections. Int Endod J. 2005;38(6):372–80. pmid:15910472
- 59. Waltimo T, Trope M, Haapasalo M, Orstavik D. Clinical efficacy of treatment procedures in endodontic infection control and one year follow-up of periapical healing. J Endod. 2005;31(12):863–6. pmid:16306819
- 60. Ercan E. DM, Yavuz I., Özekinci T. Investigation of Microorganisms in Infected Dental Root Canals. Biotechnol Biotechnol Equip. 2006;20(2):166–72.
- 61. Ashraf H, Samiee M, Eslami G, Ghodse Hosseini MR. Presence of Candida Albicans in Root Canal System of Teeth Requiring Endodontic Retreatment with and without Periapical Lesions. Iran Endod J. 2007;2(1):24–8. pmid:24348654
- 62. Cogulu D, Uzel A, Oncag O, Eronat C. PCR-based identification of selected pathogens associated with endodontic infections in deciduous and permanent teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;106(3):443–9. pmid:18547832
- 63. Rocas IN, Hulsmann M, Siqueira JF Jr. Microorganisms in root canal-treated teeth from a German population. J Endod. 2008;34(8):926–31. pmid:18634922
- 64. Miranda TT, Vianna CR, Rodrigues L, Monteiro AS, Rosa CA, Correa A Jr. Diversity and frequency of yeasts from the dorsum of the tongue and necrotic root canals associated with primary apical periodontitis. Int Endod J. 2009;42(9):839–44. pmid:19712195
- 65. Mindere A, Kundzina R, Nikolajeva V, Eze D, Petrina Z. Microflora of root filled teeth with apical periodontitis in Latvian patients. Stomatologija. 2010;12(4):116–21. pmid:21266836
- 66. Nastri N, Nastri M, Jewtuchowicz V, Mujica M, Lovanniti C, Gualtieri A, et al. Prevalence of Candida species in necrotic pulp with chronic periapical processes. Acta Odontol Latinoam. 2011;24(2):183–7. pmid:22165317
- 67. Rocas IN, Siqueira JF Jr. Comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study. J Endod. 2011;37(2):143–50. pmid:21238793
- 68. Rocas IN, Siqueira JF Jr. In vivo antimicrobial effects of endodontic treatment procedures as assessed by molecular microbiologic techniques. J Endod. 2011;37(3):304–10. pmid:21329812
- 69. Anderson AC, Hellwig E, Vespermann R, Wittmer A, Schmid M, Karygianni L, et al. Comprehensive analysis of secondary dental root canal infections: a combination of culture and culture-independent approaches reveals new insights. PLoS One. 2012;7(11):e49576. pmid:23152922
- 70. Brito LC, Sobrinho AP, Teles RP, Socransky SS, Haffajee AD, Vieira LQ, et al. Microbiologic profile of endodontic infections from HIV- and HIV+ patients using multiple-displacement amplification and checkerboard DNA-DNA hybridization. Oral Dis. 2012;18(6):558–67. pmid:22335194
- 71. Dumani A, Yoldas O, Yilmaz S, Koksal F, Kayar B, Akcimen B, et al. Polymerase chain reaction of enterococcus faecalis and candida albicans in apical periodontitis from Turkish patients. J Clin Exp Dent. 2012;4(1):e34–9. pmid:24558522
- 72. Paiva SS, Siqueira JF Jr., Rocas IN, Carmo FL, Ferreira DC, Curvelo JA, et al. Supplementing the antimicrobial effects of chemomechanical debridement with either passive ultrasonic irrigation or a final rinse with chlorhexidine: a clinical study. J Endod. 2012;38(9):1202–6. pmid:22892736
- 73. Poptani B, Sharaff M, Archana G, Parekh V. Detection of Enterococcus faecalis and Candida albicans in previously root-filled teeth in a population of Gujarat with polymerase chain reaction. Contemp Clin Dent. 2013;4(1):62–6. pmid:23853454
- 74. Kaur A SP, Singh Soodan K, Priyadarshni P. Evaluation of prevalence of Candida species in the root canals and oral cavity of children and adult patients. IOSR J Dent Med Sci. 2014;13(7):100–4.
- 75. Karygianni L, Anderson AC, Tennert C, Kollmar K, Altenburger MJ, Hellwig E, et al. Supplementary sampling of obturation materials enhances microbial analysis of endodontic treatment failures: a proof of principle study. Clin Oral Investig. 2015;19(2):319–27. pmid:24677170
- 76. Lysakowska ME, Ciebiada-Adamiec A, Sienkiewicz M, Sokolowski J, Banaszek K. The cultivable microbiota of primary and secondary infected root canals, their susceptibility to antibiotics and association with the signs and symptoms of infection. Int Endod J. 2016;49(5):422–30. pmid:26011084
- 77. Pourhajibagher M, Ghorbanzadeh R, Parker S, Chiniforush N, Bahador A. The evaluation of cultivable microbiota profile in patients with secondary endodontic infection before and after photo-activated disinfection. Photodiagnosis Photodyn Ther. 2017;18:198–203. pmid:28249746
- 78. de Miranda RG, Colombo APV. Clinical and microbiological effectiveness of photodynamic therapy on primary endodontic infections: a 6-month randomized clinical trial. Clin Oral Investig. 2018;22(4):1751–61. pmid:29116494
- 79. Bernal-Treviño A G-AA, Méndez González V, Pozos-Guillen A. Frecuencia de Candida en conductos radiculares de dientes con infección endodóntica primaria y persistente. Rev Iberoam Micol. 2018;35(2):78–82. pmid:29605495
- 80. De la Torre-Luna R, Dominguez-Perez RA, Guillen-Nepita AL, Ayala-Herrera JL, Martinez-Martinez RE, Romero-Ayala ME, et al. Prevalence of Candida albicans in primary endodontic infections associated with a higher frequency of apical periodontitis in type two diabetes mellitus patients. Eur J Clin Microbiol Infect Dis. 2020;39(1):131–8. pmid:31529305
- 81. Zargar N, Marashi MA, Ashraf H, Hakopian R, Beigi P. Identification of microorganisms in persistent/secondary endodontic infections with respect to clinical and radiographic findings: bacterial culture and molecular detection. Iran J Microbiol. 2019;11(2):120–8. pmid:31341566
- 82. Zargar N, Ashraf H, Marashi SMA, Sabeti M, Aziz A. Identification of microorganisms in irreversible pulpitis and primary endodontic infections with respect to clinical and radiographic findings. Clin Oral Investig. 2020;24(6):2099–108. pmid:32377864
- 83. Teles AM, Manso MC, Pina C, Cabeda J. A review of microbiological root canal sampling: updating an emerging picture. Arch Oral Res. 2013;9(1):41–59.
- 84. Jackson F, Halder A. Incidence of yeasts in root canals during therapy. Br Dent J. 1963;115:459–60.
- 85. Wilson MI, Hall J. Incidence of yeasts in root canals. J Br Endod Soc. 1968;2(4):56–9. pmid:4975082
- 86. Waltimo TM, Siren EK, Torkko HL, Olsen I, Haapasalo MP. Fungi in therapy-resistant apical periodontitis. Int Endod J. 1997;30(2):96–101. pmid:10332243
- 87. Luo G, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. J Clin Microbiol. 2002;40(8):2860–5. pmid:12149343
- 88. Al-Sakati H, Kowollik S, Gabris S, Balasiu A, Ommerborn M, Pfeffer K, et al. The benefit of culture-independent methods to detect bacteria and fungi in re-infected root filled teeth: a pilot study. Int Endod J. 2021;54(1):74–84. pmid:32894604
- 89. Patel R. A Moldy Application of MALDI: MALDI-ToF Mass Spectrometry for Fungal Identification. J Fungi. 2019;5(1). pmid:30609833