The effect of subinhibitory concetrations of gentian violet on germ tube formation by candida albicans and its adherence to oral epithelial cells

Abstract

Background: Oral candidiasis is mostly caused by Candida albicans and occurs in immunocompromised individuals. The virulence factors of C. albicans include adherence to the oral epithelial cells or prosthesis, germ tube formation and hydrolytic enzyme production. It can be treated with antifungal agents and antimicrobial mouthrinses. Affordable and effective mouth rinses including gentian violet offer viable alternatives in developing countries. Studies have shown that gentian violet has antifungal properties both in vivo and in vitro. However, it is difficult to maintain the antifungal or inhibitory concentration in the oral cavity due to the diluting effect of the saliva. The effect of these sub-inhibitory concentrations has not been established. Therefore, the aim of this study was to investigate the effect of sub-inhibitory concentrations of gentian violet on the germ tube formation by C. albicans and its adherence ability to the oral epithelial cells. Methods: Ten strains of C. albicans isolated from each of the denture wearers, healthy individuals and HIV positive patients were used in the study. Gentian violet (0.5%) was purchased from the local pharmacy. Antifungal property was determined using a microdilution technique. Minimum fungicidal concentrations were recorded. Sub-inhibitory concentration (0.000244%) of gentian violet was selected and its effect on the adherence ability was investigated using well described laboratory techniques. Briefly, gentian violet exposed C. albicans cells were incubated with oral epithelial cells. Epithelial cells were harvested, glass slides were prepared, stained with the Gram stain and viewed under the microscope. The number of adherent yeast cells per 100 epithelial cells was measured. The effect of five sub-inhibitory concentrations, including the MFC, on germ tube formation was determined using a well described technique. Briefly, C. albicans cells were exposed to the various concentrations of gentian violet in the presence of horse serum. Cells were harvested, slides were prepared, stained with crystal violet and viewed under the microscope. Germ tube formation in 100 yeast cells was recorded. In both the experiments, water was used as a control. The results were compared to the control using the Kruskal-wallis test. Results: The minimum fungicidal concentrations ranged from 0.000122% to 0.000977% of gentian violet solution. Sub-inhibitory concentrations of gentian violet reduced the adherence ability of C. albicans significantly (p<0.01) compared to the control. For the strains isolated from denture wearers, normal healthy individuals and HIV positive patients, the reduction in adherence was 55%, 56% and 56% respectively. The sub-inhibitory concentrations of GV significantly (p<0.01) inhibited germ tube formation compared to the controls. The inhibition was concentration dependent, with a decreased effect on germ tube formation as the gentian violet became diluted. Up to 98% reduction was noted at the concentration of 0.000244% gentian violet. Conclusion: At high concentrations, gentian violet completely killed all C. albicans and at low concentrations it can inhibit the adherence of C. albicans cells to oral epithelial cells and inhibit the hyphae formation. These results suggest that the beneficial effects of gentian violet would last beyond the fungicidal concentrations in the treatment of candidiasis. Therefore, gentian violet can be used as a topical antifungal agent in patients with candidiasis.