Rose Bengal staining - diagnostic aid for potentially malignant and malignant disorders: A pilot study.

Background: The word "Cancer" itself is sufficient to cause a fear in the minds of people. Early detection of oral potentially malignant and malignant disorders is still a diagnostic challenge for most of the clinicians. In the present study, we have evaluated the efficacy of Rose Bengal staining in the detection of oral premalignant and malignant lesions. Aim: The aim of the study was to evaluate the efficacy of the Rose Bengal staining for the identification of dysplastic areas clinically, and then correlating the findings with the histological grading of dysplasia. Methods and Materials: A total of 20 patients were selected for the study. Patients had been evaluated clinically and histopathologically along with the grading of dysplasia, and Rose Bengal stain was painted over the lesional mucosa with the patient's consent. Incisional biopsies were taken from the stained area of the Rose Bengal dye, and studied histopathologically. Grading of dysplasia and intensity of the Rose Bengal staining were found to be correlated. Chi square test was performed and a statistical significance of P<0.001 was observed. Statistical significance was defined as P<0.001. Results and Conclusion: Grading of dysplasia and the intensity of Rose Bengal staining were directly proportional to each other according to this study. P value was found to be significant. Thus Rose Bengal stain can be used as a diagnostic aid in the detection of oral potentially malignant and malignant disorders.

Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis.

Context: Single cell gel electrophoresis (SCGE) or "comet assay" is a rapid and very sensitive fluorescent microscopic method for detecting various forms of DNA damage at individual cell level. Aims: The aim of the present study was to detect the extent of DNA damage in oral cancer, oral submucous fibrosis (OSMF) and leukoplakia in comparison to normal individual. Settings and Design: A total of 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls) were recruited from Dr. R. Ahmed Dental College and Hospital and were assessed for the extent of DNA damage using SCGE following clinical diagnosis. Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA). Pearson's product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe's pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD) tail lengths (in mm) for cancer (24.95 ± 5.09) and leukoplakia (12.96 ± 2.68) were significantly greater than in controls (8.54 ± 2.55, P<0.05). After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92), the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77) was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001). Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage.

Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis.

Context and Aims: The oral cavity is the most predominant location in the head and neck region for primary malignant epithelial tumors. Oral cancer is estimated to be the sixth most common malignancy. Early recognition is imperative for successful treatment and good prognosis. Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological specimen. Materials and Methods: Smears were collected from 20 individuals with oral lesions suspicious of malignancy, oral lesions not suggestive of malignancy and normal buccal mucosa. One smear was stained with Papanicolaou stain and another one with acridine orange stain. The differences in the study group and control group were compared by means of the χ2 (Chi-square) test. The results were considered statistically significant whenever P was <0.05. Results: The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. However, the acridine orange fluorescence stain did not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content. Conclusion: The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases.

Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer.

Purpose: Telomerase is a specialized ribonucleoprotein complex that stabilizes telomeres by adding "TAG" repeats to the end of chromosomes. The catalytic subunit of telomerase is human telomerase reverse transcriptase (hTERT), whose expression is the critical determinant of telomerase activity. Telomeres and telomerases play an important role in the longevity of cell and are known to conform "immortalization" on neoplastic cells. Although there exists a lot of information on telomerase in oral cancer, very little is known about their expression in leukoplakia and oral submucous fibrosis (OSF). This study addresses this lacuna. Materials and Methods: In this preliminary study, immunohistochemistry (IHC) was used to detect the expression of hTERT protein in oral squamous cell carcinoma (OSCC) (n=30), leukoplakia (n=15), OSF (n=15) and normal oral mucosa (n=10). The cellular localization of immunostain, intensity of stain, mean nuclear labeling index (LI) and mean nuclear labeling score (LS) of hTERT protein were studied. A total number of 1000 cells were counted in each slide. All the data were analyzed using SPSS software version 10.0.2. The cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity and LI were compared across the groups using Pearson's χ2 test. The mean LI and LS for OSF, leukoplakia, OSCC and normal were compared using analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant. Results: The mean nuclear LI increased from OSF (22.46±4.53), through normal (28.3±12.3) to OSCC (47.56±21.30) (P=0.002) and from normal (28.3±12.3), through leukoplakia (44.06±14.6), to OSCC (47.56±21.30) (P=0.00). The mean nuclear labeling score was observed to increase from OSF (37.8±15), through normal (64.9±30.7), to OSCC samples (106.9±29.77) (P=0.00) and from normal (64.9±30.7), through leukoplakia (85.6±25.1) to OSCC samples (106.9±29.77) (P=0.00). Conclusion: There was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. The cellular localization, LI and LS in OSF were significantly different from OSCC and leukoplakia.

Microleakage of bonded amalgam restorations using different adhesive agents with dye under vacuum: An in vitro study.

Aim: In an effort to minimize tooth preparation, yet provide additional retention to compromised tooth structure, bonded amalgam restorations were introduced. Various resin-based adhesives have been tried earlier under bonded amalgam restorations. Still there are controversies regarding the outcome of bonded amalgam restorations regarding their adaptability to the tooth structure and microleakage. Therefore, this study was undertaken to compare the microleakage of bonded amalgam restorations using different adhesive materials. Materials and Methods: Standard Class I cavities were prepared on occlusal surfaces of 60 human molars. Teeth (n=60) were divided into three groups according to the material employed, as follows: group I: amalgam with glass ionomer cement (GIC) (type I); group II: amalgam with resin cement (Panavia F 2.0) and group III: amalgam with Copalex varnish as a control. Following restoration, the teeth were submitted to thermal cycling. The teeth were subsequently immersed in 2% rhodamine B dye under vacuum for 48 hours and sectioned to allow the assessment of microleakage under stereomicroscope. Results: The values were tabulated and the results were statistically analyzed using analysis of variance (ANOVA), Tukey's post hoc test and Kruskal-Wallis test. Amalgam with type I GIC showed the least leakage with no statistically significant difference (P value 0.226) when compared to amalgam with Panavia F 2.0 and amalgam with varnish (P value 0.107). Conclusion: It can be concluded that bonded amalgam with type I GIC is a good alternative to amalgam with resin cement (Panavia F 2.0) and amalgam with varnish for large restorations, with the added advantages of GICs. Clinical Significance: Bonded amalgam restorations prevent over-preparation and reduce the tooth flexure. GIC type I under amalgam provides chemical bonding in between amalgam and tooth structure and thus reduces the microleakage.

Morphology of collagen fibers and elastic system fibers in actinic cheilitis.

Background: Actinic cheilitis (AC) is a premalignant condition intimately related to exposure of the lips to sun rays. Aim: The objective of this study was to evaluate the elastic and collagen fibers in the lamina propria of AC. The degree of epithelial atypia was correlated with the quantity of elastic and collagen fibers. Materials and Methods: Fifty-one cases were investigated. One slide was stained with hematoxylin-eosin for the evaluation of atypia, the second was stained with Weigert's resorcin-fuchsin for the assessment of elastic fibers, and the third slide was stained with Mallory's trichrome for the analysis of collagen fibers. Results: Ordinal logistic regression analysis revealed a significant correlation between the presence of atypia and collagen fibers (P<0.05). Conclusions: It was concluded that there seems to be a reduction in the quantity of collagen fibers in cases of moderate and severe atypia. No correlation was observed between the degradation of elastic system fibers and the grade of dysplasia.

Pathosis associated with radiographically normal follicular tissues in third molar impactions: a clinicopathological study.

BACKGROUND: The follicular tissue around impacted third molars has a potential to develop pathosis. However, it is generally assumed that the absence of abnormal radiolucency indicates the presence of a normal follicle. AIMS: The aim of this study was to investigate abnormalities associated with radiographically normal follicular tissue of third molar impactions. MATERIALS AND METHODS: One hundred eighty-five impacted third molars from 170 patients with no signs of abnormal radiolucency (follicular space < 3 mm) were used for this study. Follicular tissues of the relevant teeth were collected. Specimens were fixed in 10% formalin and stained routinely with hematoxilin and eosin to be independently examined by two pathologists. A diagnosis was registered only when the results from both pathologists were in concordance. Clinical details for each patient were registered in WHO standard forms to undergo chi-square statistical analysis. RESULTS: Fifty-three per cent of the specimens had developed pathosis. The incidence of pathosis was higher in the age group of 20-30 years, in men compared to women and in the mandible compared to the maxilla. CONCLUSION: The findings of this study suggest that radiographic appearance may not be reliable in the diagnosis of pathosis in follicular tissue as a surprisingly high rate of pathosis was found in the absence of any radiographically detectable sign.

Retention and penetration of a conventional resin-based sealant and a photochromatic flowable composite resin placed on occlusal pits and fissures.

This study compares the retention and penetration of a conventional resin-based sealant (Fluroshield) and a photochromatic flowable composite resin (Tetric Flow Chroma) placed on occlusal pits and fissures and submitted to thermal or chemical cycling regimens. Penetration assessment--ten premolars were sealed with each material, isolated (except for the sealed surface) and immersed in 0.2% Rhodamine B. The teeth were serially sectioned in a mesiodistal direction. The images of the sections were digitized and analyzed (ImageLab). The distance between the most superficial and the deepest points on the occlusal central groove was calculated to determine the groove's total depth. The length of the central groove filled with the sealant was divided by its total depth to obtain the percentage of sealing of the occlusal groove. Retention assessment--30 premolars were sealed, their occlusal surfaces were photographed and the area occupied by the sealing materials was demarcated (ImageLab). The teeth were submitted to different treatments: thermocycled, stored in artificial saliva and immersed in acetic acid and saliva (10 cycles/day protocol for 30 days). New photographs were taken to assess the final area occupied by the materials. The difference between the final and initial area was calculated to obtain the material loss. The data was analyzed (two-way ANOVA and Tukey's test P<0.05). Both materials presented similar penetration of the occlusal central groove. After thermal and chemical cycling, the materials did not differ with respect to retention, except for immersion in acetic acid. In this case, Tetric Flow Chroma presented greater retention than Fluoroshield.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. AIM OF THE STUDY: 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. MATERIALS AND METHODS: Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. RESULTS: A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). CONCLUSION: One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

Brucellosis: the first case of King Chulalongkorn Memorial Hospital and review of the literature.

Brucellosis remains a major zoonotic disease worldwide. It has never been reported at King Chulalongkorn Memorial Hospital (KCMH). The authors describe the first case of brucellosis in KCMH, and also review all previous reports in Thailand. The presented case was a 52-year-old Thai man, living in Phetchabun Province, who was diagnosed with idiopathic pulmonary fibrosis two years prior to admission. He presented with prolonged fever, dry cough, weight loss of eight kg over three months, hepatosplenomegaly, and pancytopenia. Blood and bone marrow cultures grew Brucella melitensis at 72 hours of incubation. A slide agglutination (Rose Bengal) test was also positive for Brucella antibody. He had been exposed to contaminated placenta of his goats that had spontaneous abortion in the past few months before his illness. The patient was successfully treated with gentamicin, doxycycline, and rifampicin. Clinicians should have a high index of suspicion when evaluating patients presenting with prolonged fever and having an exposure risk of brucellosis.

Immunohistochemical evaluation of expression of cytokeratin 19 in different histological grades of leukoplakia and oral squamous cell carcinoma.

Recent progress in understanding the biology of keratins together with the development of monoclonal antibodies to individual keratin proteins provide the foundation for studying the keratin expression in normal and pathological oral epithelia. Cytokeratin (CK) alterations have been reported in carcinomas and these have been associated with specific aspects of tumour behaviour. Immunohisto-chemistry with monospecific CK19 antibody was used to study the expression pattern in normal mucosa, dysplasias, and oral squamous cell carcinomas (OSCC). In non-keratinzed normal mucosa, CK19 was detected in the basal cell layer, while in dysplasias (diagnosed in H and E stained sections, mild-severe) stained strongly for CK 19 in the basal and supra basal cell layers indicating layer specificity for CK 19 expression. In OSCC, in the number of CK19 labelled cells increased from well to poorly differentiated tumour. Thus the results of the present study indicate an alteration in synthesis of keratin proteins when exposed to carcinogens.

Cytological study of copper in oral submucous fibrosis.

BACKGROUND: Habitual arecanut chewing is associated with Oral Submucous Fibrosis (OSF). High copper content in arecanut plays a vital role in pathogenesis of OSF. This study evaluates the copper-staining pattern of buccal epithelial cells in oral cytological smears of non-chewers, chewers, and OSF. MATERIALS & METHODS: Alcohol fixed buccal epithelial smears of 10 histopathologically confirmed cases of OSF, 10 chewers without lesion, and 5 nonchewers were stained with modified Rhodamine technique and studied under the light microscope. Buccal epithelial smears of non-chewers dipped in copper sulphate solution were used as known positive for copper. RESULT: Copper appeared as shades of palered within the cytoplasm of chewers and did not show any stain in non-chewers. Intense red stain was seen in OSF smears as dark granules within the cytoplasm. CONCLUSION: Intense staining of copper in OSF buccal smears, than in the chewers supports the role of copper in the pathogenesis of OSF.

Seroprevalence of brucellosis in man.

A total of 352 human serum samples were screened for brucellosis. A combination of serological tests including Rose Bengal plate test (RBPT), standard tube agglutination test (STAT) and dot-enzyme linked immunosorbent assay (dot-ELISA) were employed for the purpose. The study revealed a prevalence rate of 4.97 per cent in samples that included specimens from persons occupationally exposed to animals. The number of seropositives through all tests used was higher among males (5.95 per cent) than females (3.15 per cent). A markedly higher prevalence of 17.39 per cent was recorded among field veterinarians. A low prevalence (2-6 per cent) was observed in humans with unknown history of animal contact. Dot-ELISA yielded 4.97 per cent positives compared to 1.38 and 0.82 per cent through RBPT and STAT respectively.

Determination of the morphological irregularities in the middle and apical 1/3rd region of the root canal system of permanent maxillary incisors.

A thorough knowledge of the root canal anatomy and an understanding of its variations from the normal are mandatory for the successful root canal therapy. The assessment and exploration of the accessory canal and apical delta is necessary to combat the persistent infection at the periapical area. The purpose of this study is to determine the morphological irregularities in the middle and apical 1/3rd region of the root canal system of maxillary incisors. A total of 100 maxillary incisors were decalcified, processed, sectioned at the middle and apical 1/3rd region and observed under an ordinary microscope. The frequency of accessory canals, apical delta and type of canal configuration were studied. Accessory canals were found in 5% of the teeth. There was absence of apical delta in all the specimens. Total specimens showed single canal extended from the pulp chamber to the apex (Type 1 canal configuration).

Laser and visible light cured pit and fissure sealants--comparison of microleakage at enamel-sealant interface: an in vitro study.

The efficacy of Argon laser in comparison with Visible light was assessed by the extent of marginal leakage of three commercially available pit and fissure sealants at enamel sealant interface. Enameloplasty and penetration by Rhodamine dye revealed higher degree of microleakage in Argon laser cured samples. Concise Sealant (3M) showed lesser amount of microleakage in both the curing techniques.

Evaluation of sputum staining by modified cold method and comparison with Ziehl-Neelsen and fluorochrome methods for the primary diagnosis of tuberculosis.

An improved acid-fast staining technique for sputum examination for the primary diagnosis of tuberculosis is described. The technique was modified and simplified by the elimination of heating and by combining the stages of counterstaining: making the technique easier and safer, with less risk of phenol aerosols. The efficiency of this method was evaluated by comparison with two conventional methods, Ziehl-Neelsen (ZN) staining and fluorochrome staining; culture was deemed the gold standard for tuberculosis diagnosis. Of the 392 sputum samples examined, 22.7%, 19.4% and 22.9% were positive by the ZN, fluorochrome and modified cold (MC) staining methods respectively. In comparison with culture results, the sensitivities of ZN, fluorochrome and MC methods were 68.9%, 59.7% and 70.6% respectively; the specificities were 97.4%, 98.2% and 97.8% respectively and the efficiencies were 88.8%, 86.5% and 89.5% respectively. The fluorochrome method was statistically less sensitive than the ZN and MC (p < 0.05), but no significant differences between the ZN and MC were found (p > 0.05). The results of the MC and ZN methods were in close agreement (97.2%); the slides stained by these techniques could be stored for a long time and the staining reagents were stable for several weeks. In conclusion, the MC method proved to be a valuable alternative to ZN staining for the primary diagnosis of tuberculosis.

A comparative evaluation of four adhesive tooth coloured restorative materials. An invitro study.

Four adhesive tooth colored restorative materials, Fuji II, Fuji II LC, Dyract and TPH composite resin were evaluated for the amount of microleakage in enamel and cementum. The TPH composite resin showed maximum microleakage, significantly higher than Fuji II, which showed least microleakage. All the four materials showed more micro leakage in cementum when compared to enamel.

Detection of Mycobacterium tuberculosis from sputum collected on filter paper and stored at room temperature for 5 days by PCR assay and culture.

The efficacy of PCR assay and culture for direct detection of M. tuberculosis (MTB) from sputum specimens collected on filter paper and stored at room temperature for 5 days was evaluated in comparison with conventional culture of fresh sputum specimen. A total of 231 sputum specimens were examined. MTB was recovered from 124 samples by culture from fresh sputum samples before storage. The culture positivity rate was significantly decreased to 70 per cent after 5 day's storage on filter paper. For PCR assay, a fragment of 377-bp of the IS6110 sequence was amplified and detected using nested PCR. Compared with culture results performed on fresh sputum samples, the sensitivity, specificity, and efficiency for the nested PCR were 96.0, 97.2 and 96.5 per cent, respectively. The nested PCR showed sensitivity and specificity with no significant difference (p>0.05) from culture of fresh sputum specimens. CONCLUSION: The collection and storage of sputum on filter paper at room temperature for 5 days had no apparent effect on the performance of nested PCR. Sputum samples collected by this method could be sent by post in a minimum of space and inexpensive way and will enable a large number of samples collected in the field or from peripheral health centers to be sent to central laboratories for analysis by trained technicians and under a well-equipped and well-established quality control system. The rapid and reliable detection by PCR-based assay will be helpful for optimal patient management of therapy and effective control of tuberculosis.