The early diagnosis of oral lesions, particularly potentially malignant disorders (PMDs) and oral cancers, remains a cornerstone in effective patient management and improved survival rates. Globally, oral cancer continues to be a major public health issue, especially in developing nations where tobacco use in various forms is rampant. Despite advancements in diagnostic modalities, a significant proportion of oral cancers are still diagnosed at advanced stages. This delay in detection is often due to the lack of accessible, affordable, and effective screening tools that can aid in distinguishing benign from malignant or pre-malignant lesions in the oral cavity [1].

Visual and tactile clinical examination, though widely used, is inherently subjective and heavily dependent on the examiner's experience. To enhance diagnostic accuracy, several adjunctive techniques have been developed, among which vital staining using diagnostic dyes has gained considerable attention. Dyes such as toluidine blue, methylene blue, and Lugol’s iodine have been employed for their ability to selectively stain tissues based on biochemical and structural changes associated with neoplastic transformation [2]. These dyes function by binding to acidic tissue components such as nucleic acids, thereby highlighting areas of high cellular turnover, a hallmark of dysplastic and neoplastic tissues.

Among the dyes, toluidine blue is perhaps the most widely studied and utilized. It is a metachromatic dye with an affinity for DNA and RNA, making it a reliable marker for increased nuclear content, which is characteristic of dysplastic or malignant cells. Similarly, methylene blue is a vital dye that interacts with anionic molecules in cells and has shown potential in screening for epithelial dysplasia and carcinoma [3]. Lugol’s iodine, on the other hand, stains glycogen-containing normal epithelium but fails to stain areas with depleted glycogen such as malignant lesions, providing a contrasting visual tool during examination [4].

The diagnostic utility of these dyes lies not only in their ability to detect lesions but also in their specificity and sensitivity. Numerous studies have attempted to validate their clinical applicability, often comparing their outcomes with histopathological findings, which remain the gold standard. However, the results vary across populations and settings, highlighting the need for further comparative research. Some dyes may yield false positives due to inflammation or trauma, while others may miss early dysplastic changes due to their staining mechanism. Therefore, a systematic evaluation of multiple dyes on a variety of oral lesions is essential to understand their diagnostic performance [5].

This comparative study aims to assess and evaluate the effectiveness of different dyes in diagnosing various types of oral lesions, including PMDs and oral cancers. By comparing staining patterns, sensitivity, specificity, and correlation with histopathological outcomes, this research seeks to provide evidence-based recommendations for the clinical use of diagnostic dyes. Such findings could guide clinicians in selecting appropriate adjunctive diagnostic aids in resource-constrained environments and help bridge the gap between suspicion and definitive diagnosis [6].

Ultimately, enhancing early detection through reliable staining techniques may lead to prompt intervention, reduced treatment morbidity, and improved prognosis. As non-invasive and cost-effective tools, vital dyes could play a significant role in community-based screening programs and routine dental practice [7]. This study explores these possibilities while highlighting the strengths and limitations of each dye evaluated [8–10].

Study Design and Setting

This prospective, cross-sectional comparative study was conducte at a tertiary care centre over a period of 12 months. Ethical approval was obtained from the Institutional Ethical Committee prior to commencement of the study. Informed written consent was taken from all participants.

Sample Size and Selection Criteria

A total of 90 patients presenting with clinically visible oral lesions were recruited. Patients were randomly assigned into three groups (n=30 each), with each group undergoing examination using a different diagnostic dye: toluidine blue, methylene blue, and Lugol’s iodine. Inclusion criteria were: patients aged 18 years and above, having visible oral mucosal lesions suspected to be potentially malignant disorders (PMDs) or early carcinomas, and willing to undergo biopsy. Exclusion criteria included patients with known allergies to dyes, those undergoing treatment for oral lesions, or with systemic conditions interfering with dye uptake or wound healing.

Preparation and Application of Dyes

Each dye was prepared freshly on the day of examination using standard concentrations:

• Toluidine blue: 1% aqueous solution with acetic acid pre- and post-rinse.
• Methylene blue: 1% solution with distilled water rinse.
• Lugol’s iodine: 5% iodine and 10% potassium iodide in aqueous solution.

Before dye application, oral lesions were rinsed with saline and dried using gauze. Pre-rinse (for toluidine blue) was done with 1% acetic acid for 20 seconds. The dye was then applied using a cotton applicator directly over the lesion. After a 60-second contact period, the area was rinsed again with saline (or acetic acid for toluidine blue), and excess dye was wiped off.

Clinical Evaluation and Interpretation

Staining patterns were evaluated immediately under adequate light. A positive stain was defined as a distinct uptake of dye in the lesion compared to surrounding mucosa. Weak or diffuse staining was considered equivocal, while absence of staining was recorded as negative. Staining results were recorded photographically and compared with histopathological findings.

Histopathological Examination

Incisional biopsies were performed on all stained lesions regardless of dye outcome. Tissue specimens were fixed in 10% formalin, processed, and stained with hematoxylin and eosin (H&E). Histopathological diagnosis was classified into non-dysplastic, dysplastic, and malignant categories based on WHO classification of oral epithelial lesions.

Statistical Analysis

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each dye were calculated using histopathological diagnosis as the gold standard. Chi-square tests were used to compare staining outcomes among the three dye groups. A p-value of <0.05 was considered statistically significant. Data analysis was performed using SPSS version 26.0.

A total of 90 patients were enrolled and divided equally into three groups of 30 each, based on the diagnostic dye used (toluidine blue, methylene blue, and Lugol’s iodine). The mean age of participants was 47.2 ± 11.5 years, with a male predominance (62%). The most commonly affected site was the buccal mucosa, followed by the tongue and floor of the mouth. Lesions included leukoplakia, oral submucous fibrosis (OSMF), erythroplakia, and suspected squamous cell carcinoma (SCC). Histopathological diagnosis revealed that 18 cases (20%) were malignant, 32 (35.6%) were dysplastic, and 40 (44.4%) were non-dysplastic.

Findings (Table 1):

Toluidine blue showed the highest number of positive staining outcomes (80%), followed by methylene blue (70%), and Lugol’s iodine (56.7%). Equivocal staining was observed more frequently in Lugol’s iodine group. The rate of negative results was lowest in the toluidine blue group, suggesting better lesion affinity.

Findings (Table 2):

Toluidine blue demonstrated the highest sensitivity (92.9%) and specificity (90%), making it the most reliable dye in terms of both detecting dysplastic/malignant lesions and avoiding false positives. Methylene blue showed moderately good diagnostic values, while Lugol’s iodine had the lowest sensitivity and specificity.

Findings (Table 3):

Toluidine blue outperformed other dyes in terms of overall diagnostic accuracy (91.4%). Methylene blue followed with 85.9%, and Lugol’s iodine was the least accurate (77.1%). Positive predictive and negative predictive values followed a similar trend.

Findings (Table 4):

Buccal mucosa was the most common site of positive staining in confirmed dysplastic or malignant lesions across all dye groups. Toluidine blue showed better detection at all anatomical sites, followed by methylene blue. Lugol’s iodine had relatively lower positivity across sites.

Table 1: Distribution of Staining Results across Dye Groups

Table 2: Correlation between Dye Staining and Histopathology

Table 3: Diagnostic Accuracy of Dyes in Detecting Dysplasia or Malignancy

Table 4: Site-wise Distribution of Positive Staining in Confirmed Dysplastic/Malignant Lesions

The present study evaluated and compared the diagnostic performance of three vital dyes—toluidine blue, methylene blue, and Lugol’s iodine—in detecting dysplastic and malignant changes in oral lesions. The findings clearly highlight the value of using adjunctive diagnostic aids in improving early detection of potentially malignant disorders (PMDs) and oral cancers. Among the three dyes tested, toluidine blue showed superior diagnostic accuracy, closely followed by methylene blue, with Lugol’s iodine performing least effectively.

Toluidine blue exhibited a diagnostic accuracy of 91.4%, with high sensitivity (92.9%) and specificity (90.0%). These results align with previous studies that have underscored the utility of toluidine blue in screening for high-risk lesions. The dye binds preferentially to DNA and RNA, making it an ideal marker for identifying areas with high nuclear activity, such as dysplastic and malignant tissues [11]. In this study, toluidine blue also demonstrated the highest positive predictive value (89.7%) and negative predictive value (93.1%), reinforcing its reliability in both identifying true positives and excluding false negatives.

Methylene blue performed moderately well, with an overall diagnostic accuracy of 85.9%. It achieved a sensitivity of 85.2% and specificity of 86.7%, indicating its potential usefulness, particularly in resource-limited settings where toluidine blue might not be available. While methylene blue does not offer metachromasia, it interacts with cellular components through ionic mechanisms and stains nuclei-rich areas effectively. Its slightly lower performance compared to toluidine blue may be due to its weaker binding affinity and greater variability in tissue uptake [12].

Lugol’s iodine, on the other hand, showed the least sensitivity (75%) and specificity (80%), which limited its utility as a standalone diagnostic tool. Lugol’s iodine functions differently by binding to glycogen-rich normal cells and leaving dysplastic and malignant tissues unstained due to glycogen depletion. This inverse staining mechanism may lead to higher rates of equivocal or false-negative results, particularly in lesions with partial glycogen retention [13]. In this study, Lugol’s iodine showed the highest proportion of equivocal results (16.7%), which can contribute to clinical uncertainty during screening.

Site-wise analysis revealed that the buccal mucosa was the most commonly affected site, consistent with the high prevalence of tobacco chewing in the study population. All dyes demonstrated higher staining positivity for buccal mucosa lesions compared to other sites like the tongue or floor of the mouth. This may be attributed to greater lesion accessibility, wider lesion area, and more pronounced dysplastic changes in these regions [14].

A major strength of this study is the use of histopathological diagnosis as the gold standard, which allowed for objective assessment of the dye’s performance. The findings reinforce the value of using vital dyes in conjunction with clinical examination to improve early detection, especially in primary care settings where access to biopsy may be delayed or limited. Non-invasive, affordable, and quick, these dyes offer a practical solution for screening large populations and identifying high-risk lesions for further investigation [15].

However, certain limitations must be acknowledged. First, false positives were observed with all dyes, particularly in inflamed or ulcerated lesions where cellular turnover is naturally high. This could potentially result in over-referral or patient anxiety. Second, some lesions showed equivocal staining, raising concerns about the subjectivity in dye interpretation. Standardized protocols and training may help mitigate this variability [16]. Third, dye-based screening cannot replace histopathology, but should be viewed as an adjunct to clinical judgment and biopsy.

The findings of this study also support integrating vital staining methods into public health screening programs. In areas where high oral cancer burden exists but biopsy access is constrained, such tools may bridge the gap between suspicion and confirmation. Toluidine blue, in particular, with its high diagnostic accuracy, may be incorporated into routine dental examinations or community outreach efforts [17]. Methylene blue, given its similar efficacy and availability, can serve as a suitable alternative. Lugol’s iodine, while less effective alone, might still have a role when combined with other diagnostic aids [18].

Future research should aim at combining multiple dyes or integrating dye staining with adjunctive tools like autofluorescence or optical coherence tomography to enhance diagnostic accuracy. Longitudinal studies can also help assess the prognostic value of dye uptake patterns in predicting lesion progression [19]. Additionally, studies comparing staining in immunocompromised versus immunocompetent populations, or evaluating cost-effectiveness, could further refine clinical protocols [20].

In conclusion, this comparative study demonstrates that toluidine blue is a reliable and effective diagnostic dye for detecting dysplastic and malignant oral lesions. Methylene blue offers moderate diagnostic value, while Lugol’s iodine shows lower accuracy but may still aid clinical evaluation. The use of vital dyes as adjuncts in oral cancer screening can play a significant role in enhancing early diagnosis, particularly in high-risk, resource-limited populations.

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