Oral microbiome in Proliferative Verrucous Leukoplakia exhibits loss of diversity and enrichment of pathogens.

OBJECTIVES: Oral microbiome plays an important role in oral diseases. Among them, proliferative verrucous leucoplakia (PVL) is an uncommon form of progressive multifocal leukoplakia with a worryingly rate of malignant transformation. Here, we aimed to characterize the oral microbiome of PVL patients and compare it with those of healthy controls. MATERIAL AND METHODS: Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their microbial communities. The sequence of the V3-V4 region of 16S rRNA gene was used as the taxonomic basis to estimate and analyze the composition and diversity of bacterial populations present in the samples. RESULTS: Our results show that the oral microbial composition and diversity are significantly different among PVL patients and healthy donors. The average number of observed operational taxonomic units (OTUs) was higher for healthy donors than for PVL, proving a loss of diversity in PVL. Several OTUs were found to be more abundant in either group. Among those that were significantly enriched in PVL patients, potential protumorigenic pathogens like Oribacterium sp. oral taxon 108, Campylobacter jejuni, uncultured Eubacterium sp., Tannerella, and Porphyromonas were identified. CONCLUSION: Oral microbiome dysbiosis was found in patients suffering from PVL. To the best of our knowledge, this is the first study investigating the oral microbiome alterations in PVL and, due to the limited number of participants, additional studies are needed. Oral microbiota-based biomarkers may be helpful in predicting the risks for the development of PVL.

Feasibility of investigating the association between bacterial pathogens and oral leukoplakia in low and middle income countries: A population-based pilot study in India.

BACKGROUND: Certain oral bacterial pathogens may play a role in oral carcinogenesis. We assessed the feasibility of conducting a population-based study in India to examine the distributions and levels of Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia in relation to oral leukoplakia (a potentially malignant disorder) and other participant characteristics. METHODS: This exploratory case-control study was nested within a large urban Indian cohort and the data included 22 men and women with oral leukoplakia (cases) and 69 leukoplakia-free controls. Each participant provided a salivary rinse sample, and a subset of 34 participants (9 cases; 25 controls) also provided a gingival swab sample from keratinized gingival surface for quantitative polymerase chain reaction (qPCR). RESULTS: Neither the distribution nor the levels of pathogens were associated with oral leukoplakia; however, individual pathogen levels were more strongly correlated with each other in cases compared to controls. Among controls, the median level of total pathogens was the highest (7.55×104 copies/ng DNA) among persons of low socioeconomic status. Salivary rinse provided better DNA concentration than gingival swab for qPCR analysis (mean concentration: 1.8 ng/µl vs. 0.2 ng/µl). CONCLUSIONS: This study confirms the feasibility of population studies evaluating oral microbiome in low-resource settings and identifies promising leads for future research.

Ultrastructural Changes during the Life Cycle of Mycoplasma salivarium in Oral Biopsies from Patients with Oral Leukoplakia.

Bacteria in genus Mycoplasma spp. are the smallest and simplest form of freely replicating bacteria, with 16 species known to infect humans. In the mouth, M. salivarium is the most frequently identified species. Mycoplasma spp. are parasites with small genomes. Although most of the Mycoplasma spp. that infect humans remain attached to the host cell surface throughout their life cycle, we have previously reported the presence of Mycoplasma salivarium in the epithelial cells of oral leukoplakia and oral lichen planus. However, the mechanism underlying the pathogenicity of M. salivarium has remained unclear. Further studies are needed to identify the process of infection of human cells and the stages in the life cycle of M. salivarium. Electron microscopy (EM) is the method of choice for morphological investigation of Mycoplasma spp. in cells or tissues. This study was performed to clarify and detail the ultrastructure of M. salivarium in tissue biopsies of oral mucosal leukoplakia, using three EM methods: (1) a standard EM processing method; (2) an ultracryotomy and immunolabeling method; and (3) the LR White resin post-embedding and immunolabeling method. This study included five oral leukoplakia tissue samples showing hyperplasia and hyperkeratosis. Although there was some variation in ultrastructural appearances between the three EM methods used, there were four ultrastructural appearances that are believed to reflect the stages of the M. salivarium life cycle in the epithelial cells of the oral mucosa: (1) small, electron-dense cellular-like structures or elementary bodies of M. salivarium; (2) large structures of M. salivarium; (3) M. salivarium organisms in cell division; (4) the sequence of events in the life cycle of M. salivarium that includes: (a) elementary bodies of M. salivarium deep in the oral mucosal epithelium; (b) replication by binary fission and daughter cell division from the elementary bodies; (c) maturation or degeneration of M. salivarium in the epithelial cells mainly in the upper part of the epithelium; and (d) death of the organisms in the granular and/or keratinized layer. These ultrastructural images may provide a useful reference for the identification of M. salivarium in diagnostic cytology or biopsy material.

Candida infection in oral leukoplakia: an unperceived public health problem.

OBJECTIVES: The study aimed to determine the proportion, known risk factors and etiology for Candida infection in leukoplakia lesions among patients with oral leukoplakia attending the Oral and Maxillofacial Clinic at a Tertiary Care Hospital in Sri Lanka. MATERIALS AND METHODS: Eighty clinically suspected oral leukoplakia patients were included. Two oral swabs each, from leukoplakia patients: one swab from the lesion and the other one from the contralateral unaffected corresponding area (as a control) were collected. Direct microscopy and culture followed by colony count and phenotypic identification were performed to identify pathogenic Candida species. RESULTS: Candida infection was seen in 47% of patients with oral leukoplakia. Candida albicans (94.7%) was the most common Candida species followed by Candida tropicalis (5.3%). Majority of Candida-infected lesions were seen in the buccal mucosa region. Alteration of taste (p = 0.021), having other oral lesions (p = 0.008), angular cheilitis (p = 0.024) and periodontitis (p = 0.041) showed a significant association with Candida-associated leukoplakia. Increasing age showed a significant tendency for Candida infection (p = 0.020). Smoking (p = 0.026) and betel-quid chewing (p = 0.006) were also found to be significantly associated, although alcohol consumption alone did not show a significant association. Oral leukoplakia patients who had all three habits: alcohol consumption, smoking and betel-quid chewing had a significant association with Candida infection (p = 0.004). CONCLUSIONS: Patients who had a combination of risk factors: smoking, betel-quid chewing and alcohol consumption were seen to have a significant association with Candida infection. Further betel-quid chewing alone and smoking singly was also significantly associated with Candida infection in oral leukoplakia.

The presence of Helicobacter pylori in oral cavities of patients with leukoplakia and oral lichen planus.

OBJECTIVE: Helicobacter pylori infection is one of the most common bacterial infections in men. This gastrointestinal pathogen is closely related to gastritis, peptic ulcers, and the increased risk of gastric cancer. Numerous studies have indicated oral cavities as possible Helicobacter pylori reservoirs. Helicobacter pylori has been detected both in supragingival and subgingival plaques, and also in saliva. In addition, the relationship between lesions of oral mucosa and the presence of H. pylori has been evaluated and described in some studies. The aim of this study was to assess the presence of Helicobacter pylori DNA in the oral cavity of patients with oral leukoplakia and oral lichen planus. MATERIAL AND METHODS: The study included 54 patients with oral leukoplakia, 72 with oral lichen planus lesions, and 40 healthy controls. The presence of Helicobacter pylori in oral cavity samples was analyzed using a single-step Polymerase Chain Reaction (PCR) method. All patients underwent a periodontal examination and the following clinical parameters were collected: pocket depth, bleeding, and plaque indexes. The periodontal status was assessed using the Offenbacher classification. RESULTS: In most patients, pathological lesions were in typical sites on the buccal mucosa (leukoplakia in 88%, and oral lichen planus in 93% of patients). The DNA of the Helicobacter pylori was present in 20% of patients with leukoplakia and 23% of patients with lichen planus. We did not find the DNA of H. pylori in healthy controls. The periodontal status described by periodontal indices was worse in the investigated group than in the control group. CONCLUSION: These findings suggest that the H. pylori presence in oral cavities may be related with leukoplakia and lichen planus oral lesions.

The presence of Helicobacter pylori in oral cavities of patients with leukoplakia and oral lichen planus

ABSTRACT Objective Helicobacter pylori infection is one of the most common bacterial infections in men. This gastrointestinal pathogen is closely related to gastritis, peptic ulcers, and the increased risk of gastric cancer. Numerous studies have indicated oral cavities as possible Helicobacter pylori reservoirs. Helicobacter pylori has been detected both in supragingival and subgingival plaques, and also in saliva. In addition, the relationship between lesions of oral mucosa and the presence of H. pylori has been evaluated and described in some studies. The aim of this study was to assess the presence of Helicobacter pylori DNA in the oral cavity of patients with oral leukoplakia and oral lichen planus. Material and Methods The study included 54 patients with oral leukoplakia, 72 with oral lichen planus lesions, and 40 healthy controls. The presence of Helicobacter pylori in oral cavity samples was analyzed using a single-step Polymerase Chain Reaction (PCR) method. All patients underwent a periodontal examination and the following clinical parameters were collected: pocket depth, bleeding, and plaque indexes. The periodontal status was assessed using the Offenbacher classification. Results In most patients, pathological lesions were in typical sites on the buccal mucosa (leukoplakia in 88%, and oral lichen planus in 93% of patients). The DNA of the Helicobacter pylori was present in 20% of patients with leukoplakia and 23% of patients with lichen planus. We did not find the DNA of H. pylori in healthy controls. The periodontal status described by periodontal indices was worse in the investigated group than in the control group. Conclusion These findings suggest that the H. pylori presence in oral cavities may be related with leukoplakia and lichen planus oral lesions.

In situ immunohistochemical detection of intracellular Mycoplasma salivarium in the epithelial cells of oral leukoplakia.

BACKGROUND: Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue. OBJECTIVE: Our aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry. DESIGN: We produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry. RESULTS: We obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivarium DNA in the epithelial cells of leukoplakia. CONCLUSION: Intracellular M. salivarium was identified in the epithelial cells of leukoplakia.

Clinicopathologic assessment of Candida colonization of oral leukoplakia.

BACKGROUND: Leukoplakia is the most common premalignant lesion of the oral mucosa. We studied the colonization of Candida in oral leukoplakia using direct microscopy, culture and histopathology to determine if there is a statistical correlation between Candida invasion and the clinical appearance and presence of epithelial dysplasia in leukoplakia. METHODS: Samples were collected from 40 patients with oral leukoplakia and 21 controls. The swabs collected were used to inoculate Sabouraud's dextrose agar slant and for direct microscopy with Gram's stain. Culture growths were subjected to germ tube and corn meal agar tests to differentiate between Candida albicans and non-albicans groups. Biopsies were also done in all patients for histopathological confirmation; Gomori's methanamine silver stain was used to identify fungal invasion of lesional epithelium. RESULTS AND CONCLUSIONS: Nineteen cases of leukoplakia showed Candida on direct smears, compared to 3 controls. Eighteen cases and one control showed growth of Candida on culture. Non-homogenous leukoplakia showed a higher positivity rate on microscopy and culture than homogenous lesions. All these correlations were statistically significant. Forty percent of leukoplakia cases were simultaneously positive for Candida on direct microscopy, culture and histopathologic evaluation. No significant difference was found between non-dysplastic and distinctly dysplastic lesions with respect to Candida detection on microscopy or culture.

Detection of Candida albicans ADH1 and ADH2 mRNAs in human archival oral biopsy samples.

OBJECTIVES: The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia. MATERIALS AND METHODS: Archival FFPE samples were obtained from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC), and non-CHC dysplastic leukoplakia. The presence of C. albicans was determined by periodic acid Schiff staining and by immunocytochemistry. C. albicans ADH1 and ADH2 mRNAs were detected using reverse transcription PCR. RESULTS: Candida albicans was detected in FFPE samples diagnosed as CHC (the histological diagnoses had been made by specialist oral pathologists, using uniform criteria), but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed a significant correlation between the expression of CaADH1 mRNA (P = 0.000), but not for CaADH2 mRNA (P = 0.056) in archival FFPE samples (n = 31) from biopsies of leukoplakia. CONCLUSIONS: Candida albicans was the predominant species in the lesions diagnosed as CHC, and the presence of C. albicans in CHC lesions was associated with a high expression of C. albicans ADH1 mRNA. There was no association between the presence of Candida and malignant transformation in the cases examined; however, the number of cases was limited and further studies are needed to further elucidate the role of C. albicans ADH1 in the pathogenesis of oral squamous cell carcinoma.

Mycological and histological associations of Candida in oral mucosal lesions.

The present study aimed to assess the presence and level of colonization of Candida in patients with oral mucosal lesions, to determine the presence or absence of candidal hyphae in biopsy specimens and to correlate the degree of epithelial dysplasia with the number of colony-forming units of Candida. We performed a prospective study including 50 patients diagnosed as having oral potentially malignant and malignant disorders. These patients had lesions such as leukoplakia, lichen planus, lichenoid reaction, verrucous carcinoma and oral squamous cell carcinoma. An oral swish with 10 mL of normal saline was performed, and this was collected in a sterile plastic container. Candidal colony-forming units were assessed in the specimen. This was followed by a biopsy of the lesion, which was sent for histopathologic examination for dysplasia and severity, and to assess the presence or absence of candidal hyphae. The results of the present study revealed a correlation between higher Candida colonization and increasing severity of dysplasia. An effort was made to correlate Candida by histologic and mycologic means with epithelial dysplasia. If such a correlation is strongly established, then the importance of antimycotic therapy can be emphasized to avoid deterioration.

[Integrated approach to the treatment of oral mucosa diseases in patients with chronic gastritis].

The article presents data on the clinical and microbiological short and long term efficacy of treatment of the oral mucosa diseases in patients with Helicobacter pylori associated and not associated chronic gastritis depending on the chosen treatment regimen.

Production of carcinogenic acetaldehyde by Candida albicans from patients with potentially malignant oral mucosal disorders.

OBJECTIVES: Production of carcinogenic acetaldehyde by Candida has been suggested to contribute to epithelial dysplasia and oral carcinogenesis. Oral lichen planus (OLP), oral lichenoid lesion (OLL) and oral leukoplakia (OL) are potentially carcinogenic oral diseases where colonisation by Candida is common, but acetaldehyde production by Candida has not been studied. STUDY DESIGN: Acetaldehyde production in ethanol (11 mM), glucose (100 mM), ethanol-glucose (11 mM and 100 mM) or red wine (1200 mM ethanol) incubation by Candida albicans from patients with OLL (n = 6), OLP (n = 16), OL (n = 6) and controls (n = 6) was measured by gas chromatography. Participants completed a questionnaire regarding their smoking habits and alcohol consumption. RESULTS: All Candida albicans isolates produced potentially carcinogenic levels of acetaldehyde (>100 µM) in all incubations containing ethanol. The control group isolates produced the highest acetaldehyde levels. Isolates from smokers produced more acetaldehyde in all incubations than those from non-smokers. The difference was significant in ethanol-glucose incubation. Isolates from patients who were both smokers and drinkers produced the highest amounts when incubated in ethanol, ethanol-glucose and wine. CONCLUSIONS: Candida albicans isolated from potentially carcinogenic oral diseases can produce mutagenic amounts of acetaldehyde. Cigarette smoking and alcohol consumption may favour adaptational changes resulting in the upregulation of candidal acetaldehyde metabolism.

Candidal infection in oral leukoplakia: a clinicopathologic study of 396 patients from eastern China.

Previous studies have suggested a link between the presence of Candida invasion and oral premalignant lesion. The objective of the current study was to investigate the clinicopathologic features of candidal infection in biopsies of a large retrospective cohort of patients with premalignant oral leukoplakia (n = 396) from eastern China and assess the clinical implications. Candidal hyphae were detected with periodic acid-Schiff staining of the biopsy samples. The results showed that 59 patients (15.9%) with oral leukoplakia were infected by Candida. The average age of the patients with candidal leukoplakia was 60.7 years with equal sex ratio. The tongue was the predominant site (66.1%). Epithelial hyperplasia and dysplasia were involved in 44.1% and 55.9% of patients, respectively. Multivariate analysis revealed that patient older than 60 years (odd ratio [OR], 2.28; P = .005), lesion located at the tongue (OR, 1.89; P = .038), and presence of dysplasia (OR, 2.02; P = .018) were significant risk factors of candidal infection in oral leukoplakia. Collectively, clinicopathologic features of candidal leukoplakia in eastern China were elucidated. A point to highlight was that we identified a subpopulation that was more liable to candidal infection. Elderly patients with oral tongue leukoplakia with epithelial dysplasia had much higher risk of candidal infection. Antifungal therapy was further recommended to be routine treatment of this subpopulation.

Spectrophotometric analysis of the expression of secreted aspartyl proteinases from Candida in leukoplakia and oral squamous cell carcinoma.

Candida species are a normal commensal of the oral cavity in healthy individuals, but can become an opportunistic pathogen when the oral ecosystem is unbalanced. Several virulence attributes have been identified in candidal infection, among which are the hydrolases, including the secreted aspartyl proteinases (Saps). This study evaluated and compared the in vitro level of Saps from Candida albicans in nonsmokers, smokers, and patients with leukoplakia and oral squamous cell carcinoma (OSCC). Candida cell count (CCC) at 48 h was also assessed. The Sap level was measured by spectrophotometry in 38 clinical isolates of C. albicans obtained from the oral cavity of the four different groups. Culturing was done in yeast carbon base-bovine serum albumin. Speciation of Candida was performed by using a Candida identification kit, and CCC was measured by hemocytometer. Sap levels and CCC were higher in individuals with leukoplakia and OSCC than in nonsmokers or smokers (P = 0.001); however, there was no significant difference in Sap levels or CCC between smokers and nonsmokers (P = 0.529). Further, an intragroup correlation between CCC and Sap level was also observed. The higher level of Saps from C. albicans in individuals with leukoplakia and OSCC suggests that this pathogen plays a role in disease development and could aid in identifying the pathogenic commensal.

Detection of Candida by calcofluor white.

OBJECTIVE: To assess the efficacy of calcofluor white (CFW) is compared to Gram stain and periodic acid-Schiff in detection of Candida in oral precancer and cancer. STUDY DESIGN: The study group consisted of patients with precancer (n = 45), cancer (n = 45) and control group (n = 45). Presence of Candida was confirmed by culture inoculation along with a germ tube and carbohydrate fermentation test. The cytopathologic smears were analyzed by Papanicolaou-CFW and Gram staining, whereas, tissue sections were stained by periodic acid-Schiff and CFW staining. RESULTS: Candida albicans was the predominant species identified. A highly significant association of Candida was seen more often in cancer than in precancer. Both in cytology and histopathology Candida detection by CFW was higher. In precancer it was 48.88% in smears and 40% in tissue sections, whereas, in cancer 60% in smears and 55.55% in histopathology. CONCLUSION: Among the various diagnostic tools used in the present study, the use of CFW is seen to be a simple, effective, rapid and reliable method, both in cytopathology and histopathology.

[Oral erythroplakia and erythroleukoplakia: red and red-white dysplastic lesions of the oral mucosa--part 2: cytodiagnosis, pathogenesis, therapy, and prognostic aspects]. / Erythroplakie und Erythroleukoplakie: Rote und rot-weisse Risikoläsionen der Mundhöhlenschleimhaut. Teil 2: Aktuelle Aspekte zur Zytodiagnostik, Pathogenese, Therapie und Prognose.

The second part of the present review article presents and discusses the current literature regarding cytodiagnostic aspects, pathogenesis, therapy, incidence of recurrence, and malignant transformation rate of oral erythroplakia (OE) and oral erythroleukoplakia (OEL). Oral cytopathology, eventually in combination with DNA cytometry, can add valuable information to conventional histopathology, but is not able yet to replace the aforementioned. Numerous molecular genetic variants have been studied in precancerous lesions to gain knowledge about the prognosis of these lesions. Still, there are no evidence-based parameters available to safely detect precursor lesions that will undergo malignant transformation in the future. Excision of OE and OEL should be performed with a margin of safety using the CO2 laser or a scalpel. Data about incidence of recurrence and malignant tranformation rates of OE are mostly based upon case reports or case series. The OEL has a significantly higher risk of malignant transformation than oral leukoplakias.

Liquen plano oral (I). Aspectos clínicos etiopatogénicos y epidemiológicos / Oral lichen planus (I) clinical, etiopathobenic and epidemiologycals aspect

Se presenta un estudio clínico, etiopatogénico y epidemiológico de una muestra amplia de pacientes con liquen plano oral evaluados en la Facultad de Odontología de la Universidad Complutense de Madrid (AU)

An etiopathogenic and epidemiological clinical study conducted in a broad group of patients with oral lichen plans was evaluated at the Faculty of Dentistry at the Complutense University of Madrid (AU)

Chronic hyperplastic candidosis/candidiasis (candidal leukoplakia).

Chronic hyperplastic candidosis/candidiasis (CHC; syn. candidal leukoplakia) is a variant of oral candidosis that typically presents as a white patch on the commissures of the oral mucosa. The major etiologic agent of the disease is the oral fungal pathogen Candida predominantly belonging to Candida albicans, although other systemic co-factors, such as vitamin deficiency and generalized immune suppression, may play a contributory role. Clinically, the lesions are symptomless and regress after appropriate antifungal therapy and correction of underlying nutritional or other deficiencies. If the lesions are untreated, a minor proportion may demonstrate dysplasia and develop into carcinomas. This review outlines the demographic features, etiopathogenesis, immunological features, histopathology, and the role of Candida in the disease process. In the final part of the review, newer molecular biological aspects of the disease are considered together with the management protocols that are currently available, and directions for future research.

[Role of microflora in diseases of the buccal mucosa]. / Rol' mikroflory v patologii slizistoi obolochki rta.

The role of microflora was studied in patients with diseases of the buccal mucosa (aphthous stomatitis, lichen planus, leukoplasia, desquamative glossitis, etc.). Quantitative and qualitative composition of the microflora and its sensitivity to antibiotics, antifungal agents, and bacteriophages were studied. The following changes in the microflora were distinguished: dysbiotic shift, dysbacteriosis of the I-II, III, and IV degree. Microbiological status of patients with diseases of the buccal mucosa is essential for the course, outcome, and prognosis of the underlying diseases.