Evaluation of Presence of Enterococcus faecalis in Root Canals of Deciduous Molars with Necrotic Pulp by Agar Culture and Polymerase Chain Reaction.

Objective: To assess the presence of Enterococcus faecalis in root canals of deciduous molars with necrotic pulp by agar culture and polymerase chain reaction (PCR) assay. Materials and methods: This is an experimental study, where a total of 120 endodontic samples were taken from deciduous molars with necrotic pulps. The presence of Enterococcus faecalis was assessed by culture, using Enterococcus confirmatory agar, and by PCR assay. Statistical analysis of the results was performed using McNemar's test. Results: The presence of Enterococcus faecalis was detected in 20 samples (16.67% of total) by microbial culture and in 45 samples (37.5% of total) by PCR assay, with a statistically significant difference between the two methods (p < 0.001). Microbial culture and PCR both detect Enterococcus faecalis, with the latter detecting an additional 25 positive samples. Conclusion: In this study, PCR assay was significantly more sensitive than agar culture method in detecting the presence of Enterococcus faecalis in root canals of deciduous molars with necrotic pulp, that is, 37.5% of all samples. Clinical significance: Importance of presence of Enterococcus faecalis in necrotic pulps of deciduous teeth, as it is primarily responsible for failure of endodontic treatment, thus helping clinicians to advocate the use of local drug delivery in primary teeth endodontics and also aids clinicians in choosing the most effective intracanal medication. How to cite this article: Nalawade TM, Bhat KG, Kale AD, et al. Evaluation of Presence of Enterococcus faecalis in Root Canals of Deciduous Molars with Necrotic Pulp by Agar Culture and Polymerase Chain Reaction. Int J Clin Pediatr Dent 2023;16(6):816-819.

Isolation, culture and characterization of primary cell lines of human buccal mucosal fibroblasts: A combination of explant enzamytic technique.

BACKGROUND: The cell culture technique has become a routine and a popular method for its wide applications in the field of cell biology and biotechnology and in medical research. Isolation of primary cells over the cancer cells is an essential component of cell culture technology as they are the reliable source to understand normal physiological, morphological and molecular process of human cells. As fibroblasts are the prominent cells of the connective tissue of oral mucosa, many disease entities and histogenesis are linked to fibroblasts. Culture of oral fibroblast cells helps the oral biologists and researchers to study the morphological and molecular process in the oral diseases. AIM: The aim of our experiment is to isolate and culture the human buccal mucosal fibroblast cells from healthy individuals using a combination of explant-enzymatic method and characterization of the cells by short tandem repeat (STR) profiling. MATERIALS AND METHODS: The tissue samples were collected from healthy individuals undergoing routine impacted third molar extraction. A combination of explant-enzymatic technique was used for the isolation from the tissue samples. The cells were further subcultured, maintained and stored as per the standard protocols. Thus, to confirm the oral fibroblasts of human origin and its uniqueness, they were characterized using STR profiling. RESULTS AND CONCLUSION: Using the combination technique, we were successful in isolating the cells at a faster rate by detachment of cells on day 3 and confluency on day 10. The morphological assessment and STR profiling further confirmed that the isolated cell lines resemble human fibroblast cells.

Roles of the matricellular protein Tenascin-C in T-lymphocyte trafficking and etiopathogenesis of Oral Lichen Planus.

OBJECTIVE: This study was aimed at examining the role of Tenascin-C in T cell trafficking in Oral Lichen Planus (OLP). DESIGN: For the in vivo immunohistochemical analyses, 115 OLP samples were collected from patients and immunostaining was performed. The intensity and distribution of TN-C expression were quantified and correlated with histological analyses of basement membrane integrity and presence of inflammatory infiltrate. For the in vitro study, TN-C and collagen were coated on culture plates and migration of T lymphocytes was assessed. RESULTS: TN-C immunoexpression was increased in terms of both distribution and intensity along the basement membrane zone. These changes were significantly associated with basement membrane duplication (distribution p < 0.002 and intensity p < 0.001) and bands of inflammation (distribution p < 0.002 and intensity p < 0.001) assessed by Chi-square test. T lymphocytes demonstrated significant migration towards TN-C as compared to collagen (n = 3, p < 0.05). CONCLUSIONS: These findings indicate TN-C may have a key role in promoting T cell migration at the epithelial-mesenchymal junction in OLP. These observations suggest TN-C could be a good target for therapeutic intervention, either in itself or synergistically with anti-inflammatory directed strategies in this chronic disease management.

Role of immunoreactive patterns of lymph nodes in neck dissection cases of oral squamous cell carcinoma: a clinical and histopathological study.

OBJECTIVES: Metastasis in oral squamous cell carcinoma (OSCC) can occur in a variety of ways, and draining lymphatics and lymph nodes serve as a common route. Prior to metastasis, lymph nodes elicit an immune response to either wall off or create a favorable environment for homing of tumor cells. This immune response to tumor stimuli is visualized by recognizing various immunoreactive patterns exhibited by the lymph node. The present study aims to evaluate the role of immuno-morphologic patterns of the lymph node in neck dissection for cases of OSCC. MATERIALS AND METHODS: Our retrospective study included 50 neck dissection cases of OSCC and a total of 1,078 lymph nodes. The grades of primary tumors with eight different immunoreactive patterns were compared. Vascularity and metastasis in lymph nodes were also evaluated. RESULTS: The lymphocyte predominant pattern was the most common immunoreactive pattern found in 396 of 1,078 lymph nodes. Patterns of lymphocyte predominant (P=0.0005), sinus histiocytosis (P=0.0500), paracortical hyperplasia (P=0.0001), cortical hyperplasia (P=0.0001), and increased vascularity (P=0.0190) were significantly associated with tumor grade. CONCLUSION: The present study adds to the understanding of lymph node immunoreactivity patterns and their correlation with tumor grade. We recommend further study of lymph node patterns for all sentinel lymph node biopsies and routine neck dissections for OSCCs.

Stature estimation from regression analysis of facial anthropometry in Indian population.

BACKGROUND: Stature or body height is one of the most important and useful anthropometric parameters which determines the physical identity of an individual. As craniofacial structures have the advantage of being composed largely of hard tissue which is relatively indestructible, the careful study of these can enable reliable determination of stature of the person in life. Studies pertaining to stature estimation from facial measurements are limited in an Indian population. The present investigation attempts to estimate stature from anthropometric dimensions of face. MATERIALS AND METHODS: The material for the present study comprises 361 Indian students (151 males and 210 females) in the age range of 21-45 years. Stature and six facial measurements were taken on each participant following standard methods and techniques. Karl Pearson's correlation coefficient and linear regression were done to estimate stature. RESULTS: The results indicate that facial measurements are strongly and positively correlated (P < 0.001) with stature. The accuracy of the computed equations was further tested on 50 randomly selected study participants of each group, which shows close approximation of actual and estimated stature. CONCLUSION: Within the limits of this study, we conclude that facial dimensions can be used as a supplementary approach for the estimation of stature but with caution, as these are population-specific approach.

Assessment of morphological parameters associated with neural invasion in oral squamous cell carcinoma.

AIMS: Neural invasion (NI) is a form of tumor spread distinct from lymphatic and vascular invasion. It has been correlated with aggressive behavior, disease recurrence and increased morbidity and mortality. Despite the importance of NI as a prognostic indicator, the mechanism and associated factors are poorly understood. Hence, the aim of this study was to determine morphological parameters associated with NI in oral squamous cell carcinoma (OSCC). METHODOLOGY: One hundred and five archival specimens of patients with primary OSCC who underwent surgical excision and radical neck dissection were included in the study. The presence of NI was analyzed in slides and correlated with clinical as well as morphological parameters using univariate and multivariate analysis. RESULTS: NI was identified in 31 cases (29%) of OSCC. NI was significantly associated with tobacco habit, lymphovascular invasion and positive surgical margins. Multivariate analysis further emphasized these factors to be significant risk factors for NI. Peri-NI and intra-NI significantly associated with the size of the tumor, while the distance of invasion was associated with advanced stage. CONCLUSION: NI is associated with multiple morphological parameters and its identification may have a significant impact on the management and prognostic evaluation of OSCC.

Assessment of cell block technique in head and neck pathology diagnoses: A preliminary study.

OBJECTIVE: The head and neck region is a composite site made of multiple tissue components. These tissues when affected by disease or pathology present with an array of changes in the tissue architecture and pattern. It is essential to visualize the cellular details and tissue patterns for accurate diagnosis and treatment planning. Aspiration cytology primarily makes use of the cellular details for diagnosing lesions of the head and neck. Despite the promising results, its use is still limited in certain cases of the head and neck. The reason implicated could be the indiscernible appearance of cells in the absence of tissue integrity. In this regard, cell blocks are known to facilitate the visualization of the cytomorphological as well as the tissue arrangement patterns. Thus, the present study was designed to evaluate the role of cell block cytology in the diagnosis of various lesions of the head and neck. METHODS: Odontogenic lesions, epithelial carcinomas and connective tissue pathology of the head and neck origin were included in the study (n = 45). Aspiration cytology smears and cell block diagnosis were compared with tissue biopsy diagnosis for determining their sensitivity (%) and diagnostic efficacy. RESULTS: Cell blocks showed distinct preservation of the architectural pattern. In case of fluid-filled lesions, the contents were preserved and correlated with the tissue biopsy results. The results of cell blocks were similar to that of tissue biopsy in majority of the cases (95.56%). CONCLUSION: We recommend using cell blocks as a part of routine laboratory practice for all head-neck cases.

Tumor budding is a potential histopathological marker in the prognosis of oral squamous cell carcinoma: Current status and future prospects.

Tumor budding (TB) is a histopathologically evident feature that represents a scattered pattern of invasion consisting of isolated single tumor epithelial cells or tumor cells in small clusters (up to 5 cells) seen primarily at the invasive front dispersed within the stroma for variable distance. Presence of TB has been linked with lymph node metastasis, recurrence, distant metastasis and reduced survival in numerous cancers including oral squamous cell carcinoma (OSCC). Tumor buds are also considered as histopathological markers of epithelial-mesenchymal transition which is a molecular process implicated as a hallmark for invasion and metastasis. This review gives an overview of the current evidence regarding TB assessment in OSCC and its future prospects.

Modified Alcohol-Formalin Cell Block Technique in Head and Neck Pathology Diagnosis.

OBJECTIVES: Cell block preparation is a recognized technique in histopathological diagnosis. This technique aids in maintaining an intact architecture and reducing the diagnostic errors associated with fine-needle aspiration cytology. Numerous techniques have been put forth, but the need for an optimal technique for routine use in the laboratory still persists. The aim of our study was to establish a cell block technique which aids in the accurate diagnosis of head and neck pathology. METHODOLOGY: A modified cell block technique was developed using alcohol-formalin as a fixative. Forty fine-needle aspiration fluids from clinically and radiologically diagnosed cases of head and neck pathology were used as samples. The cell block sections were compared with the cytology smears to determine the utility of the technique. RESULTS: The cell blocks presented with better preservation of the architectural framework and enabled a quick diagnosis. Cellular clumping was negligible, and nuclear as well as cellular details were maintained similar to tissue sections. It led to the integration of conventional techniques using 10% neutral buffered formalin with that of the 10% alcohol-formalin technique. CONCLUSION: Modified cell block technique can be used as a simple and effective tool in the routine diagnosis of head and neck pathology.

Validation of immunoexpression of tenascin-C in oral precancerous and cancerous tissues using ImageJ analysis with novel immunohistochemistry profiler plugin: An immunohistochemical quantitative analysis.

BACKGROUND: Immunohistochemistry (IHC) is a molecular technique that has grown tremendously over the years. However, the assessment is only qualitative which is subjective and causes errors. Due to this limitation, several excellent markers have not gained importance and reached clinical trials. Hence, we aimed to quantify IHC by ImageJ analysis with a novel IHC profiler plugin. ImageJ has not been tried in oral precancerous tissues with minimal attempt for matrix markers. AIM: This study aimed to validate the quantification of immunoexpression of tenascin-C (TN-C) in oral precancerous tissues and oral squamous cell carcinoma (OSCC) using ImageJ software with IHC profiler plugin. MATERIALS AND METHODS: After IHC staining for TN-C and image acquisition, ImageJ analysis was performed as per the standard recommended algorithm. Assessment was done by two observers by blinding the histopathological diagnosis. The immunoscore was assessed for interobserver variability using Kohen's kappa statistics. RESULTS: All our cases were in agreement and found to be statistically significant with P < 0.005. Moderate agreement was for mild dysplasia, moderate dysplasia and oral lichen planus. Substantial agreement was for oral submucous fibrosis and OSCC and almost perfect agreement noted for cases of severe dysplasia. CONCLUSION: IHC can now be quantified using freely downloadable software ImageJ analysis in oral precancerous tissues and OSCC. This software with good threshold control can quantify matrix marker such as TN-C. Hence, herewith, we propose that IHC markers should be quantified using ImageJ by our entire oral pathology fraternity so as to have a standard immunoscore for all markers.

Immunoexpression of programmed cell death 4 protein in normal oral mucosa, oral epithelial dysplasia and oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the frequently reported cancer of the head and neck. Recent studies are being conducted to evaluate the role of potential markers for diagnosing the stages of development of OSCC from normal cells. AIM: The aim of this study is to evaluate and compare the immunoexpression of programmed cell death 4 (PDCD4) protein in normal oral mucosa, oral epithelial dysplasia (OED) and OSCC. MATERIALS AND METHODS: Histologically diagnosed, formalin-fixed paraffin-embedded archived cases (n = 100) of normal mucosa (n = 10), OED (n = 60) and OSCC (n = 30) were analyzed immunohistochemically in the present retrospective study using monoclonal rabbit antihuman PDCD4. OED and squamous cell carcinoma were graded according to the World Health Organization and Broder's histological grading criteria, respectively. Clinical parameters and immunohistochemical results were analyzed by Fisher exact test using SPSS software. P <0.05 was indicative of significant differences. RESULTS: PDCD4 expression was observed in the normal oral mucosa, OED and OSCC. The maximum expression was observed in the normal oral mucosa, which reduced significantly in OED and OSCC (P = 0.017). With the increase in the transformation from normal cells to cancer cells, a shift from nuclear to cytoplasmic staining was observed indicating predominant cytoplasmic localization of stain as a feature of altered cells. CONCLUSION: The present study delineates the molecular difference between the normal, dysplastic and carcinomatous cells; and points toward the role of PDCD4 localization in the proliferation of cells. This study thus highlights the need for further research with inclusion of long follow-up period and other pathological criteria such as inflammation and microenvironment, immune status of patient and tumor stage, which could aid in the development of prospective diagnostic options.

Immunoexpression of Epithelial Mesenchymal Transition Proteins E-Cadherin, ß-Catenin, and N-Cadherin in Oral Squamous Cell Carcinoma.

Aims Epithelial mesenchymal transition (EMT) is a crucial process for acquisition of malignant phenotype, aggressiveness, and metastatic capacity in neoplasms. It is characterized by loss of epithelial markers and gain of mesenchymal markers. Studies on EMT and its potential association with the histological grading are sparse in oral squamous cell carcinoma (OSCC). This study aims to evaluate the expression of EMT-associated proteins-E-cadherin, ß-catenin, and N-cadherin-in different grades of OSCC. Methodology In all, 60 cases of OSCC further subdivided into 20 cases each of well-, moderately, and poorly differentiated OSCCs were stained immunohistochemically with E-cadherin, ß-catenin, and N-cadherin antibodies. The differences in the expression were evaluated using χ2 and Fisher exact tests, whereas Spearman's correlation was used to analyze the correlation between the markers. Results A reduced E-cadherin expression noted in 40% of the OSCCs was associated with reduced ß-catenin expression in 66.6% of the cases and increase in the expression of mesenchymal N-cadherin seen in 80% of cases. This expression pattern demonstrated a significant association with histological grades. A membrane to cytoplasmic shift of E-cadherin (73.3%) and ß-catenin (78.3%) increased with histological grade. A negative correlation was observed with the E-cadherin and N-cadherin localization, though it did not reach statistical significance. Conclusion OSCC tissues had high levels of EMT phenotype as compared with the normal oral mucosa. This phenotype was characterized by reduced E-cadherin and ß-catenin expression and overexpression of N-cadherin. Aberrant localization of the studied proteins was a hallmark for depicting EMT.

Diagnoses of 964 oral biopsies from people aged over 50 years in Karnataka State, India.

INTRODUCTION: The drastic epidemiological transition has created a huge void in evidence with regard to the health statistics of elderly population. Data are sparse on the oral mucosal conditions prevalent among the elderly Indian population. This study aimed to review the oral biopsies reported in the institute over the last 21 years and to examine the trends in geriatric oral pathologies. METHODOLOGY: A total of 964 histologically confirmed geriatric (age ranging 50-97 years) oral lesions among 4000 cases reported were retrieved from the files of Department of Oral Pathology and Microbiology of KLE VK institute, Belgaum, Karnataka commencing since 1992 till 2012. These cases were retrospectively evaluated for various parameters such as age, gender, duration, location and type of lesions. RESULTS: The geriatric oral lesions were seen to occur predominantly in men (65%), especially the sixth decade of life (47%). The most common location was the buccal mucosa (29%). The malignant neoplastic lesions accounted for the majority of the geriatric lesions (36%) followed by non-neoplastic lesions (27%), potentially malignant disorders (26%) and benign neoplasms, which formed a minor proportion of (11%). The oral squamous cell carcinoma was the single most common geriatric lesion accounted. Gender and age-wise correlation analyses among the various lesion categories showed statistical significance. CONCLUSION: The study provides valuable information about major oral pathologies in our scenario that contribute to significant morbidity and mortality among the geriatric population. It also sheds light about the diseases that need to be targeted in future Indian public health policies and initiatives for geriatric care.

Acetic acid as an adjunct vital stain in diagnosis of tobacco-associated oral lesions: A pilot study.

BACKGROUND: One of the most important risk factors for oral precancer and cancer in India is the use of tobacco. In chronic tobacco users, the mucosa may appear clinically healthy, however, changes are observed histologically. Screening of such tobacco users for an early diagnosis is, therefore, of paramount importance. Several adjunctive diagnostic modalities have been used in the past, but none has been conclusively validated as confirmative and cost-effective screening methodology. The aim of this study was to evaluate the use of 5% acetic acid as a vital staining agent in tobacco-associated oral lesions. MATERIALS AND METHODS: The study subjects were divided into two groups. Group I (n = 40) subjects with a history of chronic tobacco use and clinically apparent normal mucosa. Group II (n = 40) subjects suspected of having oral cancer, 5% acetic acid was applied to the mucosa/lesions, followed by incisional biopsy for confirmatory diagnosis. RESULTS: The sensitivity and specificity for Groups I and II were 97%, 50% and 95%, 60%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of Group I were 0.95 and 0.66. Group II showed PPV and NPV of 0.95 and 0.60. CONCLUSION: The results of this study suggest that acetic acid holds promise for future. Hence, further studies are needed to be undertaken on a large scale to assess its potential as a screening tool for high-risk individuals and oral cancer.

Tumor budding is an independent prognostic factor for prediction of lymph node metastasis in oral squamous cell carcinoma.

OBJECTIVE: Despite the enormous advances in diagnostic and management modalities of oral squamous cell carcinoma (OSCC), the mortality rates have remained stagnant with a 5-year survival rate of <50% challenging the available methods of prognostic assessment. Presence of tumor budding has been associated with aggressive behavior and is correlated with lymph node metastasis, recurrence, distant metastasis, and decreased survival in several cancers. However, the prognostic significance of this apparently simple to evaluate parameter is sparse in OSCC. METHODS: A total of 75 cases of surgically excised OSCC were analyzed for tumor budding along with other clinicopathologic parameters. Tumor budding was graded as high and low intensity based on presence and absence of ≥10 or <10 budding foci in hematoxylin and eosin-stained sections. An association between the clinicopathological parameters, lymph node metastases with the budding index was examined using univariate and multivariate analyses. RESULTS: Tumor budding was evident in 89% of cases with around 45.3% of the cases demonstrated high-intensity budding. High-intensity tumor budding was significantly associated with lymph node metastasis and depth of invasion. Multivariate analysis demonstrated that tumor budding and depth of invasion were significant independent predictors for lymph node metastasis. CONCLUSION: Tumor budding is frequently encountered histologic marker in OSCC. High-intensity tumor budding is a strong independent prognostic factor for prediction of lymph node metastasis.

Clinico-pathological study to evaluate oral lichen planus for the establishment of clinical and histopathological diagnostic criteria.

OBJECTIVE: Lichen planus and lichenoid lesions affecting the oral cavity show similar clinico-pathological features creating a diagnostic dilemma. Hence, the aim of the present study was to establish a clinical and histopathological correlation in the diagnosis of oral lichen planus, based on the modified WHO diagnostic criteria of oral lichen planus and oral lichenoid lesions proposed by Van der Meij and Van der Waal in 2003. MATERIAL AND METHOD: In the present study, 100 cases of oral lichen planus were clinically and histopathologically analyzed. Out of the 100 cases, 50 were prospective and 50 were retrospective cases. Prospective cases were collected based on the clinical diagnosis of oral lichen planus and oral lichenoid lesion. Retrospective cases were collected based on the histopathological diagnosis of oral lichen planus. Both the clinical and histopathological analyses were performed based on a proposal for a set of modified diagnostic criteria of oral lichen planus and oral lichenoid lesion. A final diagnosis of oral lichen planus was made only after the correlation of the clinical diagnosis with the histopathological diagnosis. RESULTS: The interobserver agreement among three observers for both prospective and retrospective cases in the final diagnosis of oral lichen planus was found to be "good" to "very good" indicating high reproducibility. However, the final diagnoses of true oral lichen planus after clinico-pathological correlation in prospective and retrospective study groups appeared to be 38.0% and 54.0% respectively. CONCLUSION: The results of the present study revealed mild to moderate clinico-pathological correlation in the final diagnosis of oral lichen planus for the prospective and retrospective study groups respectively.